Processing biomass and petroleum containing materials

ABSTRACT

Biomass (e.g., plant biomass, animal biomass, and municipal waste biomass) is processed to produce useful products, such as fuels. For example, systems can use feedstock materials, such as cellulosic and/or lignocellulosic materials and/or starchy materials, to produce ethanol and/or butanol, e.g., by fermentation.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 14/879,051, filedOct. 8, 2015, which is a continuation of U.S. Ser. No. 14/853,764, filedSep. 14, 2015, which is a continuation of U.S. Ser. No. 14/688,393,filed Apr. 16, 2015, which is a continuation of U.S. Ser. No.13/798,961, filed Mar. 13, 2013, now U.S. Pat. No. 9,023,183, issued onMay 5, 2015, which is a continuation of U.S. Ser. No. 13/042,692, filedMar. 8, 2011, now U.S. Pat. No. 8,454,803, issued on Jun. 4, 2013, whichis a continuation of U.S. Ser. No. 12/417,699, filed Apr. 3, 2009, nowU.S. Pat. No. 7,931,784, issued on Apr. 6, 2011, which claims priorityfrom U.S. Provisional Application Ser. Nos. 61/049,406, filed Apr. 30,2008, and 61/073,665, filed Jun. 18, 2008. The full disclosure of eachof these applications is incorporated by reference herein.

TECHNICAL FIELD

This invention relates to processing biomass and petroleum-containingmaterials.

BACKGROUND

Biomass, particularly biomass waste, is abundantly available. It wouldbe useful to derive materials and fuel, such as ethanol, from biomass.

It would also be useful to more efficiently process petroleum-containingmaterials to obtain fuels and other products.

SUMMARY

Biomass can be processed to alter its structure at one more levels. Theprocessed biomass can then be used as source of materials and fuel.

Many embodiments of this application use Natural Force™ Chemistry.Natural Force™ Chemistry methods use the controlled application andmanipulation of physical forces, such as particle beams, gravity, light,etc., to create intended structural and chemical molecular change. Inpreferred implementations, Natural Force™ Chemistry methods altermolecular structure without chemicals or microorganisms. By applying theprocesses of Nature, new useful matter can be created without harmfulenvironmental interference.

A method for changing a molecular and/or a supramolecular structure ofany biomass material includes treating the biomass material withradiation. In particular, the radiation can include particles,particularly charged particles. Charged particles include ions, such aspositively charged ions, such as protons, carbon or oxygen ions. Forexample, the charged particles typically are heavier than an electron orhave a different charge than an electron (e.g., a positron). Theradiation can be applied in an amount sufficient to change the molecularstructure and/or supramolecular structure of the biomass material. Thebiomass material can include carbohydrates or materials that includecarbohydrates, e.g., cellulosic materials, lignocellulosic materials,starchy materials, or mixtures of any biomass materials. Theradiation-treated material can be used to produce a product.

Particles having a different charge than electrons and/or particlesheavier than electrons can be utilized for the irradiation. For example,protons, helium nuclei, argon ions, silicon ions, neon ions, carbonions, phosphorus ions, oxygen ions or nitrogen ions can be utilized tomodify the structure of the biomass, e.g., breakdown the molecularweight or increase the molecular weight of the biomass. In someembodiments, heavier particles can induce higher amounts of chainscission in comparison to electrons or photons. In addition, in someinstances, positively charged particles can induce higher amounts ofchain scission than negatively charged particles due to their acidity.

A method for making and processing materials from biomass can includefunctionalizing materials with one or more desired types and amounts offunctional groups, and products made from the structurally changedmaterials. For example, many of the methods described herein can providecellulosic and/or lignocellulosic materials that have a lower molecularweight and/or crystallinity relative to a native material. Many of themethods provide materials that can be more readily utilized by a varietyof microorganisms to produce useful products, such as hydrogen, alcohols(e.g., ethanol or butanol), organic acids (e.g., acetic acid),hydrocarbons, co-products (e.g., proteins) or mixtures of any of these.

In some instances, functionalized biomass is more soluble and morereadily utilized by microorganisms in comparison to un-functionalizedbiomass. In addition, many of the functionalized materials describedherein are less prone to oxidation and can have enhanced long-termstability (e.g., oxidation in air under ambient conditions). Many of theproducts obtained, such as ethanol or n-butanol, can be utilized as afuel for powering cars, trucks, tractors, ships or trains, e.g., as aninternal combustion fuel or as a fuel cell feedstock. Many of theproducts obtained can also be utilized to power aircraft, such asplanes, e.g., having jet engines or helicopters. In addition, theproducts described herein can be utilized for electrical powergeneration, e.g., in a conventional steam generating plant or in a fuelcell plant.

In some embodiments, materials include a cellulosic or lignocellulosicmaterial.

In one aspect, the invention features methods of changing a molecularand/or a supramolecular structure of any biomass material that includepretreating the biomass material with radiation including chargedparticles (e.g., accelerated charged particles), such as those heavierthan an electron or having a different charge than an electron (e.g., apositron), to change the molecular structure and/or supramolecularstructure of the biomass material, and processing the pretreated biomassmaterial to produce a product. Prior to pretreating, the biomassmaterial can be optionally prepared by reducing one or more dimensionsof individual pieces of the biomass material. Charged particles includeions, such as positively charged ions, such as protons, carbon or oxygenions. Charged particles that are used to pretreat biomass can havevelocities of, e.g., from 0.05 c to about 0.9999 c, where c representsthe vacuum velocity of light.

In another aspect, the invention features methods of making a productfrom biomass, such as a combustible fuel or a fuel for a fuel cell, suchas ethanol, butanol, hydrogen, hydrocarbons or mixtures of any of these,that include providing a material that includes a carbohydrate, such asoligomeric and/or monomeric carbohydrates or derivative and analogsthereof, produced by a process that includes pretreating a biomassfeedstock with radiation that includes charged particles heavier than anelectron, such as ions, such as positively charged ions, such as protonsor carbon ions, optionally together with one or more other pretreatmentsselected from the group consisting of photonic radiation, sonication,pyrolysis, and oxidation, and contacting the material that includes thecarbohydrate with a microorganism, such as a blend of bacteria, havingthe ability to convert at least a portion, e.g., at least about 1, 2, 3,4, or 5 percent by weight, of the material to the product, such as thecombustible fuel.

The dose of radiation utilized depends upon the type and degree ofmodification that is desired and the kind of radiation employed. Forexample, to break down structures with electrons can require, e.g.,greater than about 10 MRad, whereas protons, which are more massive thanan electron and can deliver a larger effective dose, may require only 1MRad.

In one aspect, the invention features a method that includes exposing abiomass material to charged particles having a mass greater than orequal to the mass of a proton, wherein exposing the biomass materialcomprises directing the charged particles to pass through a fluid, andthen directing the charged particles to be incident on the biomassmaterial.

In some implementations the fluid is selected from the group consistingof air, oxygen, hydrogen, and reactive gases.

In another aspect, the invention features a method of treating biomass,the method including: forming a plurality of negatively charged ions,and accelerating the negatively charged ions to a first energy; removinga plurality of electrons from at least some of the negatively chargedions to form positively charged ions; accelerating the positivelycharged ions to a second energy, and directing the positively chargedions to be incident on the biomass.

Some embodiments may include one or more of the following features.Removing the plurality of electrons from at least some of the negativelycharged ions can include directing the negatively charged ions to beincident on a metal foil. Accelerating the negatively charged ions to afirst energy can include directing the ions to pass through a pluralityof electrodes at different electrostatic potentials. The method canfurther include altering trajectories of the positively charged ionsbefore the ions are accelerated to the second energy.

In yet another aspect, the invention features a method of treatingbiomass that includes generating a plurality of charged particles;accelerating the plurality of charged particles by directing each of thecharged particles to make multiple passes through an accelerator cavitycomprising a time-dependent electric field; and exposing the biomass tothe accelerated charged particles.

Some embodiments may include one or more of the following features. Anorientation of the electric field can be selected to correspond to adirection of motion of the charged particles in the accelerator cavity.

In a further aspect, the invention features a method of treating biomassthat includes generating a plurality of charged particles; acceleratingthe plurality of charged particles by directing the charged particles topass through an acceleration cavity comprising multiple electrodes atdifferent potentials; and exposing the biomass to the acceleratedcharged particles.

In another aspect, the invention features a method of treating biomassthat includes generating a plurality of charged particles; acceleratingthe plurality of charged particles by directing the charged particles topass through an accelerator comprising multiple waveguides, wherein eachwaveguide comprises an electromagnetic field; and exposing the biomassto the accelerated charged particles.

Some embodiments include one or more of the following features. Theelectromagnetic field in each of the waveguides can be a time-varyingfield. The electromagnetic field in each of the waveguides can begenerated by a microwave field source. The electromagnetic fields ineach of the waveguides can be generated to coincide with passage of thecharged particles through each of the waveguides.

Any of the methods discussed herein can include one or more of thefollowing features. Some embodiments may include one or more of thefollowing features. The charged particles may include ions, in somecases two or more different types of ions. The charged particles may benegatively charged. The charged particles can be selected from the groupconsisting of hydrogen ions, carbon ions, oxygen ions, nitrogen ions,halogen ions, and noble gas ions. The method can further includeexposing the biomass to a plurality of electrons.

In yet another aspect, the invention features a method that includesexposing a petroleum-containing material to an ion beam, and processingthe petroleum-containing material to extract a hydrocarbon componentfrom the petroleum-containing material.

Some implementations may include one or more of the following features.The ion beam can include positively charged ions. The ion beam caninclude at least one of protons, carbon ions, oxygen ions, and noble gasions. In some cases, the ion beam includes at least one of platinumions, palladium ions, rhenium ions, iridium ions, ruthenium ions,aluminum ions, nickel ions, and osmium ions. The petroleum-containingmaterial can include crude oil, in which case the crude oil can in somecases be exposed to the ion beam before the crude oil is refined.Processing the petroleum containing material can include refining atleast a portion of the material in at least one step selected from thegroup consisting of a catalytic cracking process, a catalytic reformingprocess, a catalytic hydrocracking process, and an alkylation process.The method can further include exposing the material to an electronbeam, and/or exposing the material to a reactive gas, such as ozone,during exposure of the material to the ion beam. Exposing the materialto the ion beam can include exposing the material to a first type ofions from a first ion beam, and exposing the material from a second typeof ions from a second ion beam. In some cases, the first and secondtypes of ions have different charges, and/or different masses. Duringexposure to the ion beam the material can be flowing.

In another aspect, the invention features methods of changing amolecular and/or a supramolecular structure of a biomass feedstock thatinclude 1) irradiating the biomass feedstock with radiation, such asphotons, electrons or ions of sufficient energy to ionize the biomassfeedstock, to provide a first level of radicals; and 2) quenching theradicals to an extent that the radicals are at a second level lower thanthe first level. The irradiated biomass feedstock can be processed toproduce a product. The first level of radicals may be detectable, e.g.,with an electron spin resonance spectrometer. For example, the secondlevel can be detectably less than the first level, or a level that thatis no longer detectable with the electron spin resonance spectrometer,e.g., such as at a level of less than about 10¹⁴ spins. If desired,prior to irradiation and/or after irradiation, the biomass feedstock canbe prepared by reducing one or more dimensions of individual pieces ofthe biomass feedstock.

In another aspect, the invention features methods of making a product,such as a fuel, such as a combustible fuel, such as a motor, an aviationfuel or a fuel cell fuel, e.g., for generating electricity, that includea) providing a material that includes a carbohydrate produced by aprocess comprising 1) irradiating a biomass feedstock with ionizingradiation to provide a first level of radicals, and 2) quenching theradicals to an extent that the radicals are present at a second levellower than the first level. The material can then be contacted with amicroorganism, e.g., to convert the material, for example to a productsuch as a combustible fuel. The microorganism can have the ability toconvert at least a portion, e.g., at least about 1, 2, 3, 4, or 5percent by weight, of the biomass to the product.

Examples of biomass feedstock include paper, paper products, paperwaste, wood, particle board, sawdust, agricultural waste, sewage,silage, grasses, rice hulls, bagasse, cotton, jute, hemp, flax, bamboo,sisal, abaca, straw, corn cobs, corn stover, switchgrass, alfalfa, hay,rice hulls, coconut hair, cotton, synthetic celluloses, seaweed, algae,or mixtures of these. The biomass can be or can include a natural or asynthetic material.

Examples of fuels include one or more of hydrogen, alcohols, andhydrocarbons. For example, the alcohols can be ethanol, n-propanol,isopropanol, n-butanol, or mixtures of these.

In some examples, the biomass feedstock can be prepared by shearing abiomass fiber source to provide a fibrous material. For example, theshearing can be performed with a rotary knife cutter. The fibers of thefibrous material can have, e.g., an average length-to-diameter ratio ofgreater than 5/1. The fibrous material can have, e.g., a BET surfacearea of greater than 0.25 m²/g.

In some embodiments, the pretreated biomass material can further includea buffer, such as sodium bicarbonate or ammonium chloride, anelectrolyte, such as potassium chloride or sodium chloride a growthfactor, such as biotin and/or a base pair such as uracil, a surfactant,a mineral, or a chelating agent.

In some embodiments, the biomass is sheared, and the sheared biomass caninclude discrete fibers having a length-to-diameter ratio (L/D) ofgreater than about 5/1. For example, the biomass can have internalfibers, and the biomass has been sheared to an extent that its internalfibers are substantially exposed. For example, the biomass has beensheared to an extent that it has a bulk density of less than about 0.35g/cm³. Low bulk density materials can be deeply penetrated by chargedparticles. For example, for electrons at an average energy of 5 MeV anda material with a bulk density of 0.35 g/cm³, electron penetrationdepths can be 5-7 inches or more.

In still another aspect, the invention features a system that includes:one or more of: (1) a biomass reservoir, (2) a particle beam source(e.g., an accelerator), and (3) a delivery module for moving biomassfrom the biomass reservoir into range of the particle beam. The systemcan be designed for continuous processing of biomass. See e.g.,conveyance and processing methods described in Ser. No. 61/049,404. Incertain cases, the particle beam source provides a beam of at least 20,40, or 60 cm in length and biomass (e.g., switchgrass, stover, or otherplant waste) is located in the beam.

To further aid in the reduction of the molecular weight of thecellulose, an enzyme, e.g., a cellulolytic enzyme, or a chemical, e.g.,sodium hypochlorite, an acid, a base or a swelling agent, can beutilized with any method described herein. The enzyme and/or chemicaltreatment can occur before, during or after sonication.

In some embodiments, no chemicals, e.g., no swelling agents, are addedto the biomass prior to irradiation. For example, alkaline substances(such as sodium hydroxide, potassium hydroxide, lithium hydroxide andammonium hydroxides), acidifying agents (such as mineral acids (e.g.,sulfuric acid, hydrochloric acid and phosphoric acid)), salts, such aszinc chloride, calcium carbonate, sodium carbonate,benzyltrimethylammonium sulfate, or basic organic amines, such asethylene diamine, is added prior to irradiation or other processing. Insome cases, no additional water is added. For example, the biomass priorto processing can have less than 0.5 percent by weight added chemicals,e.g., less than 0.4, 0.25, 0.15 or 0.1 percent by weight addedchemicals. In some instances, the biomass has no more than a trace,e.g., less than 0.05 percent by weight added chemicals, prior toirradiation. In other instances, the biomass prior to irradiation hassubstantially no added chemicals or swelling agents. Avoiding the use ofsuch chemicals can also be extended throughout, e.g., at all times priorto fermentation, or at all times.

When a microorganism is utilized, it can be a natural microorganism oran engineered microorganism. For example, the microorganism can be abacterium, e.g., a cellulolytic bacterium, a fungus, e.g., a yeast, aplant or a protist, e.g., an algae, a protozoa or a fungus-like protist,e.g., a slime mold. When the organisms are compatible, mixtures may beutilized. Generally, various microorganisms can produce a number ofuseful products, such as a fuel, by operating on, e.g., fermenting thematerials. For example, alcohols, organic acids, hydrocarbons, hydrogen,proteins or mixtures of any of these materials can be produced byfermentation or other processes.

In some embodiments, the method may include passing sheared materialthrough one or more screens, e.g., a screen having an average openingsize of 1.59 mm or less (0.0625 inch). Screening separates the materialaccording to size. For example, in one embodiment, the method includes:shearing the fiber source to produce a sheared fiber source; passing thesheared fiber source through a first screen to produce a screened fibersource; shearing the screened fiber source to produce a second shearedfiber source; passing the second sheared fiber source through a secondscreen having an average opening size less than the first screen toprovide a second screened fiber source; and steam exploding the secondscreened fiber source to produce the fibrous material. The method mayfurther include shearing the second screened fiber source to produce athird sheared fiber source, and then steam exploding the third shearedfibers source to produce the fibrous material.

It is also possible to shear the fiber source and concurrently pass itthrough a screen.

The methods may also further include encapsulating the fibrous materialin a substantially gas impermeable material to remove entrapped gas anddensify the fibrous material. The substantially gas impermeable materialmay be soluble in water, and may be provided in the form of a bag.

Examples of products that may be produced include mono- andpolyfunctional C1-C6 alkyl alcohols, mono- and poly-functionalcarboxylic acids, C1-C6 hydrocarbons, and combinations thereof. Specificexamples of suitable alcohols include methanol, ethanol, propanol,isopropanol, butanol, ethylene glycol, propylene glycol, 1,4-butanediol, glycerin, and combinations thereof. Specific example of suitablecarboxylic acids include formic acid, acetic acid, propionic acid,butyric acid, valeric acid, caproic acid, palmitic acid, stearic acid,oxalic acid, malonic acid, succinic acid, glutaric acid, oleic acid,linoleic acid, glycolic acid, lactic acid, γ-hydroxybutyric acid, andcombinations thereof. Examples of suitable hydrocarbons include methane,ethane, propane, pentane, n-hexane, and combinations thereof. Many ofthese products may be used as fuels.

The term “fibrous material,” as used herein, is a material that includesnumerous loose, discrete and separable fibers. For example, a fibrousmaterial can be prepared from a bleached Kraft paper fiber source byshearing, e.g., with a rotary knife cutter.

The term “screen,” as used herein, means a member capable of sievingmaterial according to size. Examples of screens include a perforatedplate, cylinder or the like, or a wire mesh or cloth fabric.

The term “pyrolysis,” as used herein, means to break bonds in a materialby the application of heat energy. Pyrolysis can occur while the subjectmaterial is under vacuum, or immersed in a gaseous material, such as anoxidizing gas, e.g., air or oxygen, or a reducing gas, such as hydrogen.

Oxygen content is measured by elemental analysis by pyrolyzing a samplein a furnace operating at 1300° C. or above.

For the purposes of this disclosure, carbohydrates are materials thatare composed entirely of one or more saccharide units or that includeone or more saccharide units. The saccharide units can be functionalizedabout the ring with one or more functional groups, such as carboxylicacid groups, amino groups, nitro groups, nitroso groups or nitrilegroups and still be considered carbohydrates. Carbohydrates can bepolymeric (e.g., equal to or greater than 10-mer, 100-mer, 1,000-mer,10,000-mer, or 100,000-mer), oligomeric (e.g., equal to or greater thana 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or 10-mer), trimeric,dimeric, or monomeric. When the carbohydrates are formed of more than asingle repeat unit, each repeat unit can be the same or different.

Examples of polymeric carbohydrates include cellulose, xylan, pectin,and starch, while cellobiose and lactose are examples of dimericcarbohydrates. Examples of monomeric carbohydrates include glucose andxylose.

Carbohydrates can be part of a supramolecular structure, e.g.,covalently bonded into the structure. Examples of such materials includelignocellulosic materials, such as that found in wood.

A starchy material is one that is or includes significant amounts ofstarch or a starch derivative, such as greater than about 5 percent byweight starch or starch derivative. For purposes of this disclosure, astarch is a material that is or includes an amylose, an amylopectin, ora physical and/or chemical mixture thereof, e.g., a 20:80 or 30:70percent by weight mixture of amylose to amylopectin. For example, rice,corn, and mixtures thereof are starchy materials. Starch derivativesinclude, e.g., maltodextrin, acid-modified starch, base-modified starch,bleached starch, oxidized starch, acetylated starch, acetylated andoxidized starch, phosphate-modified starch, genetically-modified starchand starch that is resistant to digestion.

For purposes of this disclosure, a low molecular weight sugar is acarbohydrate or a derivative thereof that has a formula weight(excluding moisture) that is less than about 2,000, e.g., less thanabout 1,800, 1,600, less than about 1,000, less than about 500, lessthan about 350 or less than about 250. For example, the low molecularweight sugar can be a monosaccharide, e.g., glucose or xylose, adisaccharide, e.g., cellobiose or sucrose, or a trisaccharide.

A combustible fuel is a material capable of burning in the presence ofoxygen. Examples of combustible fuels include ethanol, n-propanol,n-butanol, hydrogen and mixtures of any two or more of these.

Swelling agents as used herein are materials that cause a discernableswelling, e.g., a 2.5 percent increase in volume over an unswollen stateof cellulosic and/or lignocellulosic materials, when applied to suchmaterials as a solution, e.g., a water solution. Examples includealkaline substances, such as sodium hydroxide, potassium hydroxide,lithium hydroxide and ammonium hydroxides, acidifying agents, such asmineral acids (e.g., sulfuric acid, hydrochloric acid and phosphoricacid), salts, such as zinc chloride, calcium carbonate, sodiumcarbonate, benzyltrimethylammonium sulfate, and basic organic amines,such as ethylene diamine.

A “sheared material,” as used herein, is a material that includesdiscrete fibers in which at least about 50% of the discrete fibers, havea length/diameter (L/D) ratio of at least about 5, and that has anuncompressed bulk density of less than about 0.6 g/cm³. A shearedmaterial is thus different from a material that has been cut, chopped orground.

Changing a molecular structure of a biomass feedstock, as used herein,means to change the chemical bonding arrangement, such as the type andquantity of functional groups or conformation of the structure. Forexample, the change in the molecular structure can include changing thesupramolecular structure of the material, oxidation of the material,changing an average molecular weight, changing an average crystallinity,changing a surface area, changing a degree of polymerization, changing aporosity, changing a degree of branching, grafting on other materials,changing a crystalline domain size, or an changing an overall domainsize.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

In any of the methods disclosed herein, radiation may be applied from adevice that is in a vault.

This application incorporates by reference herein the entire contents ofInternational Application No. PCT/US2007/022719, filed Oct. 26, 2007.The full disclosures of each of the following U.S. Patent Applications,which are being filed concurrently herewith, are hereby incorporated byreference herein: Attorney Docket Nos. 08995-0062001, 08895-0063001,08895-0073001, 08895-0075001, 08895-0076001, 08895-0085001,08896-0086001, 08895-0096001, and 08895-0103001.

The entire contents of each of the following publications areincorporated herein by reference: J. R. Adney et al., IEEE Transactionson Nuclear Science, Vol. NS-32, pp. 1841-1843 (1985); J. R. Adney etal., Proceedings of the 1989 IEEE Particle Accelerator Conference, Vol.1, pp. 348-350 (1989); J. A. Ferry et al., Nuclear Instruments andMethods in Physics Research, Vol. B64, pp. 309-312 (1992); J. Ferry, inHandbook of Accelerator Physics and Engineering, pp. 16-17 (1999); J. A.Ferry et al., Nuclear Instruments and Methods in Physics Research A,Vol. 382, pp. 316-320 (1996); J. A. Ferry, Nuclear Instruments andMethods in Physics Research A, Vol. 328, pp. 28-33 (1993); T. M. Hauseret al., Nuclear Instruments and Methods in Physics Research B, Vol. 249,pp. 932-934 (2006); R. G. Herb, in Encyclopedia of Physics, pp. 3-8(1981); R. G. Herb et al., in Encyclopedia of Applied Physics, Vol. 1,pp. 27-42 (1991); R. G. Herb, IEEE Transactions on Nuclear Science, Vol.NS-30, pp. 1359-1362 (1983); R. G. Herb, Proceedings of the ThirdInternational Conference on Electrostatic Accelerator Technology (1981);G. M. Klody et al., Nuclear Instruments and Methods in Physics ResearchB, Vol. 56-57, pp. 704-707 (1991); G. M. Klody et al., NuclearInstruments and Methods in Physics Research B, Vol. 240, pp. 463-467(2005); R. L. Loger, Application of Accelerators in Research andIndustry, Proceedings of the Fifteenth International Conference, pp.640-643 (1999); G. A. Norton et al., Nuclear Instruments and Methods inPhysics Research B, Vol. 40-41, pp. 785-789 (1989); G. A. Norton et al.,Application of Accelerators in Research and Industry, Proceedings of theFourteenth International Conference, pp. 1109-1114 (1997); G Norton etal., Handbook of Accelerator Physics and Engineering, pp. 24-26 (1999);GA. Norton et al., Symposium of North Eastern Accelerator Personnel, pp.295-301 (1992); G. Norton, Pramana, Vol. 59, pp. 745-751 (2002); G. A.Norton et al., Nuclear Instruments and Methods in Physics Research B,Vol. 37-38, pp. 403-407 (1989); G. A. Norton, Heavy Ion AcceleratorTechnology: Eighth International Conference, pp. 3-23 (1999); J. E.Raatz et al., Nuclear Instruments and Methods in Physics Research A,vol. 244, pp. 104-106 (1986); R. D. Rathmell et al., Nuclear Instrumentsand Methods in Physics Research B, vol. 56-57, pp. 1072-1075 (1991); J.B. Schroeder et al., Nuclear Instruments and Methods in Physics ResearchB, Vol. 56-57, pp. 1033-1035 (1991); J. B. Schroeder, NuclearInstruments and Methods in Physics Research B, Vol. 40-41, pp. 535-537(1989); J. B. Schroeder et al., Radiocarbon, Vol. 46 (2004); J. B.Schroeder et al., Nuclear Instruments and Methods in Physics Research B,Vol. 24-25, pp. 763-766 (1987); P. H. Stelson et al., NuclearInstruments and Methods in Physics Research A, Vol. 244, pp. 73-74(1986); M. L. Sundquist et al., Nuclear Instruments and Methods inPhysics Research B, Vol. 99, pp. 684-687 (1995); M. L. Sundquist et al.,Nuclear Instruments and Methods in Physics Research A, Vol. 287, pp.87-89 (1990); and M. L. Sundquist, Applications of Accelerators inResearch and Industry, Proceedings of the Fifteenth InternationalConference, pp. 661-664 (1999). All other patents, patent applications,and references cited herein are also incorporated by reference.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a block diagram illustrating conversion of biomass intoproducts and co-products.

FIG. 2 is block diagram illustrating conversion of a fiber source into afirst and second fibrous material.

FIG. 3 is a cross-sectional view of a rotary knife cutter.

FIG. 4 is block diagram illustrating conversion of a fiber source into afirst, second and third fibrous material.

FIG. 5 is block diagram illustrating densification of a material.

FIG. 6 is a perspective view of a pellet mill.

FIG. 7A is a densified fibrous material in pellet form.

FIG. 7B is a transverse cross-section of a hollow pellet in which acenter of the hollow is in-line with a center of the pellet.

FIG. 7C is a transverse cross-section of a hollow pellet in which acenter of the hollow is out of line with the center of the pellet.

FIG. 7D is a transverse cross-section of a tri-lobal pellet.

FIG. 8 is a block diagram illustrating a treatment sequence forprocessing feedstock.

FIG. 9 is a perspective, cut-away view of a gamma irradiator housed in aconcrete vault.

FIG. 10 is an enlarged perspective view of region R of FIG. 9.

FIG. 11A is a block diagram illustrating an electron beam irradiationfeedstock pretreatment sequence.

FIG. 11B is a schematic representation of biomass being ionized, andthen oxidized or quenched.

FIG. 12 is a schematic view of a system for sonicating a process streamof cellulosic material in a liquid medium.

FIG. 13 is a schematic view of a sonicator having two transducerscoupled to a single horn.

FIG. 14 is a block diagram illustrating a pyrolytic feedstockpretreatment system.

FIG. 15 is a cross-sectional side view of a pyrolysis chamber.

FIG. 16 is a cross-sectional side view of a pyrolysis chamber.

FIG. 17 is a cross-sectional side view of a pyrolyzer that includes aheated filament.

FIG. 18 is a schematic cross-sectional side view of a Curie-Pointpyrolyzer.

FIG. 19 is a schematic cross-sectional side view of a furnace pyrolyzer.

FIG. 20 is a schematic cross-sectional top view of a laser pyrolysisapparatus.

FIG. 21 is a schematic cross-sectional top view of a tungsten filamentflash pyrolyzer.

FIG. 22 is a block diagram illustrating an oxidative feedstockpretreatment system.

FIG. 23 is block diagram illustrating a general overview of the processof converting a fiber source into a product, e.g., ethanol.

FIG. 24 is a cross-sectional view of a steam explosion apparatus.

FIG. 25 is a schematic cross-sectional side view of a hybrid electronbeam/sonication device.

FIG. 26 is a block diagram illustrating a dry milling process for cornkernels.

FIG. 27 is a block diagram illustrating a wet milling process for cornkernels.

FIG. 28 is a scanning electron micrograph of a fibrous material producedfrom polycoated paper at 25× magnification. The fibrous material wasproduced on a rotary knife cutter utilizing a screen with ⅛ inchopenings.

FIG. 29 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was produced on a rotary knife cutter utilizing a screen with ⅛inch openings.

FIG. 30 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was twice sheared on a rotary knife cutter utilizing a screenwith 1/16 inch openings during each shearing.

FIG. 31 is a scanning electron micrograph of a fibrous material producedfrom bleached Kraft board paper at 25× magnification. The fibrousmaterial was thrice sheared on a rotary knife cutter. During the firstshearing, a ⅛ inch screen was used; during the second shearing, a 1/16inch screen was used, and during the third shearing a 1/32 inch screenwas used.

FIGS. 31A-31F are 3D AFM micrographs from the surface of fibers fromsamples P132, P132-10, P132-100, P-1e, P-30e, and P-100e, respectively.

FIG. 32 is a schematic side view of a sonication apparatus, while FIG.33 is a cross-sectional view through the processing cell of FIG. 32.

FIG. 34 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 35 and 36 are scanning electron micrographs of the fibrousmaterial of FIG. 34 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 37 is a scanning electron micrographs of the fibrous material ofFIG. 34 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 38 is a scanning electron micrographs of the fibrous material ofFIG. 34 after irradiation with 100 Mrad and sonication at 1000×magnification.

FIG. 39 is an infrared spectrum of Kraft board paper sheared on a rotaryknife cutter.

FIG. 40 is an infrared spectrum of the Kraft paper of FIG. 39 afterirradiation with 100 Mrad of gamma radiation.

FIGS. 40-1 to 40-4 are infrared spectra of A, A-50e, S-50e, and S-100e,respectively.

FIGS. 40A-40I are ¹H-NMR spectra of samples P132, P132-10, P132-100,P-1e, P-5e, P-10e, P-30e, P-70e, and P-100e. FIG. 40J is a comparison ofthe exchangeable proton at ˜16 ppm from FIGS. 40A-40I. FIG. 40K is a¹³C-NMR of sample P-100e.

FIGS. 40L-40M are ¹³C-NMR of sample P-100e with a delay time of 10seconds. FIG. 40N is a ¹H-NMR at a concentration of 10% wt./wt. ofsample P-100e.

FIG. 40O is a titration curve of sample P-30e using a potentiometer.

FIG. 41 is a schematic view of a process for biomass conversion.

FIG. 42 is schematic view of another process for biomass conversion.

FIG. 43 is a schematic diagram of a field ionization source.

FIG. 44 is a schematic diagram of an electrostatic ion separator.

FIG. 45 is a schematic diagram of a field ionization generator.

FIG. 46 is a schematic diagram of a thermionic emission source.

FIG. 47 is a schematic diagram of a microwave discharge ion source.

FIG. 48 is a schematic diagram of a DC accelerator.

FIG. 49 is a schematic diagram of a recirculating accelerator.

FIG. 50 is a schematic diagram of a static accelerator.

FIG. 51 is a schematic diagram of a dynamic linear accelerator.

FIG. 52 is a schematic diagram of a van de Graaff accelerator.

FIG. 53 is a schematic diagram of a folded tandem accelerator.

FIG. 54 is a schematic diagram showing dose profiles for ions,electrons, and photons in a condensed-phase material.

FIG. 55 is a schematic diagram of an ion beam exposure system.

FIGS. 56A and 56B are schematic diagrams showing ion beam energydistributions.

FIG. 56C is a schematic diagram showing ion dose profiles in an exposedsample.

FIG. 57 is a schematic diagram of a scattering element that includesmultiple sub-regions.

FIG. 58 is a schematic diagram of an ion beam exposure system thatincludes an ion filter.

FIGS. 59A-C are schematic diagrams showing energy distributions forunfiltered and filtered ion beams.

FIG. 60 is a schematic diagram showing three ion dose profilescorresponding to exposure of a sample to ion beams having differentaverage energies.

FIG. 61A is a schematic diagram showing a net ion dose profile for anexposed sample based on the three ion dose profiles of FIG. 60.

FIG. 61B is a schematic diagram showing three different ion doseprofiles corresponding to ion beams of different average energy and ioncurrent.

FIG. 61C is a schematic diagram showing a net ion dose profile based onthe three ion dose profiles of FIG. 61B.

FIG. 62A is a schematic diagram showing three different ion doseprofiles corresponding to exposure of a sample to beams of threedifferent types of ions.

FIG. 62B is a schematic diagram showing a net ion dose profile based onthe three ion dose profiles of FIG. 62A.

FIG. 63 is a schematic diagram of a truck-based mobile biomassprocessing facility.

FIG. 64 is a schematic diagram of a train-based mobile biomassprocessing facility.

FIG. 65 is a chart showing glucose concentration (top 4 producers) inExample 29.

FIG. 65A is a chart showing cell concentrations for Z. mobilis inExample 31.

FIG. 65B is a chart showing ethanol concentrations for Z. mobilis inExample 31.

FIG. 65C is a chart showing % growth and ethanol production for Z.mobilis in Example 31.

FIG. 66 is a chart showing cell concentrations for P. stipitus inExample 31.

FIG. 66A is a chart showing ethanol concentrations for P. stipitus inExample 31.

FIG. 66B is a chart showing % growth and ethanol production for P.stipitus in Example 31.

FIG. 67 is a chart showing cell concentrations for S. cerevisiae inExample 31.

FIG. 67A is a chart showing ethanol concentrations for S. cerevisiae inExample 31.

FIG. 67B is a chart showing % growth and ethanol production for S.cerevisiae in Example 31.

DETAILED DESCRIPTION

Biomass (e.g., plant biomass, such as those that are or that include oneor more low molecular weight sugars, animal biomass, and municipal wastebiomass) can be processed to produce useful products such as fuels,e.g., fuels for internal combustion engines, jet engines or feedstocksfor fuel cells. In addition, functionalized materials having desiredtypes and amounts of functionality, such as carboxylic acid groups, enolgroups, aldehyde groups, ketone groups, nitrile groups, nitro groups, ornitroso groups, can be prepared using the methods described herein. Suchfunctionalized materials can be, e.g., more soluble, easier to utilizeby various microorganisms or can be more stable over the long term,e.g., less prone to oxidation. Systems and processes are describedherein that can use various biomass materials, such as cellulosicmaterials, lignocellulosic materials, starchy materials or materialsthat are or that include low molecular weight sugars, as feedstockmaterials. Such materials are often readily available, but can bedifficult to process, e.g., by fermentation, or can give sub-optimalyields at a slow rate, Feedstock materials are first physically preparedfor processing, often by size reduction of raw feedstock materials.Physically prepared feedstock can be pretreated or processed using oneor more of radiation, sonication, oxidation, pyrolysis, and steamexplosion. The various pretreatment systems and methods can be used incombinations of two, three, or even four of these technologies.

In some cases, to provide materials that include a carbohydrate, such ascellulose, that can be converted by a microorganism to a number ofdesirable products, such as a combustible fuels (e.g., ethanol, butanolor hydrogen), feedstocks that include one or more saccharide units canbe treated by any one or more of the processes described herein. Otherproducts and co-products that can be produced include, for example,human food, animal feed, pharmaceuticals, and nutriceuticals.

Types of Biomass

Generally, any biomass material that is or includes carbohydratescomposed entirely of one or more saccharide units or that include one ormore saccharide units can be processed by any of the methods describedherein. For example, the biomass material can be cellulosic orlignocellulosic materials, or starchy materials, such as kernels ofcorn, grains of rice or other foods, or materials that are or thatinclude one or more low molecular weight sugars, such as sucrose orcellobiose.

For example, such materials can include paper, paper products, wood,wood-related materials, particle board, grasses, rice hulls, bagasse,cotton, jute, hemp, flax, bamboo, sisal, abaca, straw, corn cobs, ricehulls, coconut hair, algae, seaweed, cotton, synthetic celluloses, ormixtures of any of these. Suitable materials include those listed in theSummary section, above.

Fiber sources include cellulosic fiber sources, including paper andpaper products (e.g., polycoated paper and Kraft paper), andlignocellulosic fiber sources, including wood, and wood-relatedmaterials, e.g., particle board. Other suitable fiber sources includenatural fiber sources, e.g., grasses, rice hulls, bagasse, cotton, jute,hemp, flax, bamboo, sisal, abaca, straw, corn cobs, rice hulls, coconuthair; fiber sources high in α-cellulose content, e.g., cotton; andsynthetic fiber sources, e.g., extruded yarn (oriented yarn orun-oriented yarn). Natural or synthetic fiber sources can be obtainedfrom virgin scrap textile materials, e.g., remnants or they can be postconsumer waste, e.g., rags. When paper products are used as fibersources, they can be virgin materials, e.g., scrap virgin materials, orthey can be post-consumer waste. Aside from virgin materials,post-consumer, industrial (e.g., offal), and processing waste (e.g.,effluent from paper processing) can also be used as fiber sources. Also,the fiber source can be obtained or derived from human (e.g., sewage),animal or plant wastes. Additional fiber sources have been described inU.S. Pat. Nos. 6,448,307, 6,258,876, 6,207,729, 5,973,035 and 5,952,105.

Examples of biomass include renewable, organic matter, such as plantbiomass (defined below), microbial biomass (defined below), animalbiomass (e.g., any animal by-product, animal waste, etc.) and municipalwaste biomass including any and all combinations of these biomassmaterials.

Plant biomass and lignocellulosic biomass include organic matter (woodyor non-woody) derived from plants, especially matter available on asustainable basis. Examples include biomass from agricultural or foodcrops (e.g., sugarcane, sugar beets or corn kernels) or an extracttherefrom (e.g., sugar from sugarcane and corn starch from corn),agricultural crop wastes and residues such as corn stover, wheat straw,rice straw, sugar cane bagasse, and the like. Plant biomass furtherincludes, but is not limited to, trees, woody energy crops, wood wastesand residues such as softwood forest thinnings, barky wastes, sawdust,paper and pulp industry waste streams, wood fiber, and the like.Additionally grass crops, such as switchgrass and the like havepotential to be produced on a large-scale as another plant biomasssource. For urban areas, the plant biomass feedstock includes yard waste(e.g., grass clippings, leaves, tree clippings, and brush) and vegetableprocessing waste.

Lignocellulosic feedstock can be plant biomass such as, but not limitedto, non-woody plant biomass, cultivated crops, such as, but not limitedto, grasses, for example, but not limited to, C4 grasses, such asswitchgrass, cord grass, rye grass, miscanthus, reed canary grass, or acombination thereof, or sugar processing residues such as bagasse, orbeet pulp, agricultural residues, for example, soybean stover, cornstover, rice straw, rice hulls, barley straw, corn cobs, wheat straw,canola straw, rice straw, oat straw, oat hulls, corn fiber, recycledwood pulp fiber, sawdust, hardwood, for example aspen wood and sawdust,softwood, or a combination thereof. Further, the lignocellulosicfeedstock may include cellulosic waste material such as, but not limitedto, newsprint, cardboard, sawdust, and the like. Lignocellulosicfeedstock may include one species of fiber or alternatively,lignocellulosic feedstock may include a mixture of fibers that originatefrom different lignocellulosic feedstocks. Furthermore, thelignocellulosic feedstock may comprise fresh lignocellulosic feedstock,partially dried lignocellulosic feedstock, fully dried lignocellulosicfeedstock or a combination thereof.

Microbial biomass includes biomass derived from naturally occurring orgenetically modified unicellular organisms and/or multicellularorganisms, e.g., organisms from the ocean, lakes, bodies of water, e.g.,salt water or fresh water, or on land, and that contains a source ofcarbohydrate (e.g., cellulose). Microbial biomass can include, but isnot limited to, for example protists (e.g., animal (e.g., protozoa suchas flagellates, amoeboids, ciliates, and sporozoa) and plant (e.g.,algae such alveolates, chlorarachniophytes, cryptomonads, euglenids,glaucophytes, haptophytes, red algae, stramenopiles, andviridaeplantae)), seaweed, plankton (e.g., macroplankton, mesoplankton,microplankton, nanoplankton, picoplankton, and femptoplankton),phytoplankton, bacteria (e.g., gram positive bacteria, gram negativebacteria, and extremophiles), yeast and/or mixtures of these. In someinstances, microbial biomass can be obtained from natural sources, e.g.,the ocean, lakes, bodies of water, e.g., salt water or fresh water, oron land. Alternatively or in addition, microbial biomass can be obtainedfrom culture systems, e.g., large scale dry and wet culture systems.

Animal biomass includes any organic waste material such asanimal-derived waste material or excrement or human waste material orexcrement (e.g., manure and sewage).

In some embodiments, the carbohydrate is or includes a material havingone or more β-1,4-linkages and having a number average molecular weightbetween about 3,000 and 50,000. Such a carbohydrate is or includescellulose (I), which is derived from (β-glucose 1) through condensationof β(1→4)-glycosidic bonds. This linkage contrasts itself with that forα(1→4)-glycosidic bonds present in starch and other carbohydrates.

Starchy materials include starch itself, e.g., corn starch, wheatstarch, potato starch or rice starch, a derivative of starch, or amaterial that includes starch, such as an edible food product or a crop.For example, the starchy material can be arracacha, buckwheat, banana,barley, cassava, kudzu, oca, sago, sorghum, regular household potatoes,sweet potato, taro, yams, or one or more beans, such as favas, lentilsor peas. Blends of any two or more starchy materials are also starchymaterials. In particular embodiments, the starchy material is derivedfrom corn. Various corn starches and derivatives are described in “CornStarch,” Corn Refiners Association (11th Edition, 2006), the contents ofwhich are incorporated herein by reference.

Biomass materials that include low molecular weight sugars can, e.g.,include at least about 0.5 percent by weight of the low molecular sugar,e.g., at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 12.5, 25, 35, 50, 60,70, 80, 90 or even at least about 95 percent by weight of the lowmolecular weight sugar. In some instances, the biomass is composedsubstantially of the low molecular weight sugar, e.g., greater than 95percent by weight, such as 96, 97, 98, 99 or substantially 100 percentby weight of the low molecular weight sugar.

Biomass materials that include low molecular weight sugars can beagricultural products or food products, such as sugarcane and sugarbeets or an extract therefrom, e.g., juice from sugarcane, or juice fromsugar beets. Biomass materials that include low molecular weight sugarscan be substantially pure extracts, such as raw or crystallized tablesugar (sucrose). Low molecular weight sugars include sugar derivatives.For example, the low molecular weight sugars can be oligomeric (e.g.,equal to or greater than a 4-mer, 5-mer, 6-mer, 7-mer, 8-mer, 9-mer or10-mer), trimeric, dimeric, or monomeric. When the carbohydrates areformed of more than a single repeat unit, each repeat unit can be thesame or different.

Specific examples of low molecular weight sugars include cellobiose,lactose, sucrose, glucose and xylose, along with derivatives thereof. Insome instances, sugar derivatives are more rapidly dissolved in solutionor utilized by microbes to provide a useful material, such as ethanol orbutanol. Examples of such sugars and sugar derivatives are shown below.

Combinations (e.g., by themselves or in combination of any biomassmaterial, component, product, and/or co-product generated using themethods described herein) of any biomass materials described herein canbe utilized for making any of the products described herein, such asethanol. For example, blends of cellulosic materials and starchymaterials can be utilized for making any product described herein.

Systems for Treating Biomass

FIG. 1 shows a system 100 for converting biomass, particularly biomasswith significant cellulosic and lignocellulosic components and/orstarchy components, into useful products and co-products. System 100includes a feed preparation subsystem 110, a pretreatment subsystem 114,a primary process subsystem 118, and a post-processing subsystem 122.Feed preparation subsystem 110 receives biomass in its raw form,physically prepares the biomass for use as feedstock by downstreamprocesses (e.g., reduces the size of and homogenizes the biomass), andstores the biomass both in its raw and feedstock forms.

Biomass feedstock with significant cellulosic and/or lignocellulosiccomponents, or starchy components can have a high average molecularweight and crystallinity that can make processing the feedstock intouseful products (e.g., fermenting the feedstock to produce ethanol)difficult. Accordingly it is useful to pretreat biomass feedstock. Asdescribed herein, in some embodiments, the pretreatment of biomass feedstock do not use acids, bases and enzymes to process cellulosic,lignocellulosic or starchy feedstocks or only use such treatments insmall or catalytic amounts.

Pretreatment subsystem 114 receives feedstock from the feed preparationsubsystem 110 and prepares the feedstock for use in primary productionprocesses by, for example, reducing the average molecular weight andcrystallinity of the feedstock. Primary process subsystem 118 receivespretreated feedstock from pretreatment subsystem 114 and produces usefulproducts (e.g., ethanol, other alcohols, pharmaceuticals, and/or foodproducts). In some cases, the output of primary process subsystem 118 isdirectly useful but, in other cases, requires further processingprovided by post-processing subsystem 122. Post-processing subsystem 122provides further processing to product streams from primary processsystem 118 which require it (e.g., distillation and denaturation ofethanol) as well as treatment for waste streams from the othersubsystems. In some cases, the co-products of subsystems 114, 118, 122can also be directly or indirectly useful as secondary products and/orin increasing the overall efficiency of system 100. For example,post-processing subsystem 122 can produce treated water to be recycledfor use as process water in other subsystems and/or can produce burnablewaste which can be used as fuel for boilers producing steam and/orelectricity.

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 2000 to10,000 dried tons of feedstock per day depending at least in part on thetype of feedstock used. The type of feedstock can also impact plantstorage requirements with plants designed primarily for processingfeedstock whose availability varies seasonally (e.g., corn stover)requiring more on- or of-site feedstock storage than plants designed toprocess feedstock whose availability is relatively steady (e.g., wastepaper).

Physical Preparation

In some cases, methods of processing begin with a physical preparationof the feedstock, e.g., size reduction of raw feedstock materials, suchas by cutting, grinding, shearing or chopping. In some cases, thematerial can be reduced into particles using a hammermill, disk-refiner,or flaker. In some cases, loose feedstock (e.g., recycled paper, starchymaterials, or switchgrass) is prepared by shearing or shredding. Screensand/or magnets can be used to remove oversized or undesirable objectssuch as, for example, rocks or nails from the feed stream.

Feed preparation systems can be configured to produce feed streams withspecific characteristics such as, for example, specific maximum sizes,specific length-to-width, or specific surface areas ratios. As a part offeed preparation, the bulk density of feedstocks can be controlled(e.g., increased or decreased).

Size Reduction

In some embodiments, the material to be processed is in the form of afibrous material that includes fibers provided by shearing a fibersource. For example, the shearing can be performed with a rotary knifecutter.

For example, and by reference to FIG. 2, a fiber source 210 is sheared,e.g., in a rotary knife cutter, to provide a first fibrous material 212.The first fibrous material 212 is passed through a first screen 214having an average opening size of 1.59 mm or less ( 1/16 inch, 0.0625inch) to provide a second fibrous material 216. If desired, fiber sourcecan be cut prior to the shearing, e.g., with a shredder. For example,when a paper is used as the fiber source, the paper can be first cutinto strips that are, e.g., ¼- to ½-inch wide, using a shredder, e.g., acounter-rotating screw shredder, such as those manufactured by Munson(Utica, N.Y.). As an alternative to shredding, the paper can be reducedin size by cutting to a desired size using a guillotine cutter. Forexample, the guillotine cutter can be used to cut the paper into sheetsthat are, e.g., 10 inches wide by 12 inches long.

In some embodiments, the shearing of fiber source and the passing of theresulting first fibrous material through first screen are performedconcurrently. The shearing and the passing can also be performed in abatch-type process.

For example, a rotary knife cutter can be used to concurrently shear thefiber source and screen the first fibrous material. Referring to FIG. 3,a rotary knife cutter 220 includes a hopper 222 that can be loaded witha shredded fiber source 224 prepared by shredding fiber source. Shreddedfiber source is sheared between stationary blades 230 and rotatingblades 232 to provide a first fibrous material 240. First fibrousmaterial 240 passes through screen 242, and the resulting second fibrousmaterial 244 is captured in bin 250. To aid in the collection of thesecond fibrous material, the bin can have a pressure below nominalatmospheric pressure, e.g., at least 10 percent below nominalatmospheric pressure, e.g., at least 25 percent below nominalatmospheric pressure, at least 50 percent below nominal atmosphericpressure, or at least 75 percent below nominal atmospheric pressure. Insome embodiments, a vacuum source 252 is utilized to maintain the binbelow nominal atmospheric pressure.

Shearing can be advantageous for “opening up” and “stressing” thefibrous materials, making the cellulose of the materials moresusceptible to chain scission and/or reduction of crystallinity. Theopen materials can also be more susceptible to oxidation whenirradiated.

The fiber source can be sheared in a dry state, a hydrated state (e.g.,having up to ten percent by weight absorbed water), or in a wet state,e.g., having between about 10 percent and about 75 percent by weightwater. The fiber source can even be sheared while partially or fullysubmerged under a liquid, such as water, ethanol, isopropanol.

The fiber source can also be sheared in under a gas (such as a stream oratmosphere of gas other than air), e.g., oxygen or nitrogen, or steam.

Other methods of making the fibrous materials include, e.g., stonegrinding, mechanical ripping or tearing, pin grinding or air attritionmilling.

If desired, the fibrous materials can be separated, e.g., continuouslyor in batches, into fractions according to their length, width, density,material type, or some combination of these attributes. For example, forforming composites, it is often desirable to have a relatively narrowdistribution of fiber lengths.

For example, ferrous materials can be separated from any of the fibrousmaterials by passing a fibrous material that includes a ferrous materialpast a magnet, e.g., an electromagnet, and then passing the resultingfibrous material through a series of screens, each screen havingdifferent sized apertures.

The fibrous materials can also be separated, e.g., by using a highvelocity gas, e.g., air. In such an approach, the fibrous materials areseparated by drawing off different fractions, which can be characterizedphotonically, if desired. Such a separation apparatus is discussed inLindsey et al, U.S. Pat. No. 6,883,667.

The fibrous materials can irradiated immediately following theirpreparation, or they can may be dried, e.g., at approximately 105° C.for 4-18 hours, so that the moisture content is, e.g., less than about0.5% before use.

If desired, lignin can be removed from any of the fibrous materials thatinclude lignin. Also, to aid in the breakdown of the materials thatinclude the cellulose, the material can be treated prior to irradiationwith heat, a chemical (e.g., mineral acid, base or a strong oxidizersuch as sodium hypochlorite) and/or an enzyme.

In some embodiments, the average opening size of the first screen isless than 0.79 mm ( 1/32 inch, 0.03125 inch), e.g., less than 0.51 mm (1/50 inch, 0.02000 inch), less than 0.40 mm ( 1/64 inch, 0.015625 inch),less than 0.23 mm (0.009 inch), less than 0.20 mm (1/128 inch, 0.0078125inch), less than 0.18 mm (0.007 inch), less than 0.13 mm (0.005 inch),or even less than less than 0.10 mm (1/256 inch, 0.00390625 inch). Thescreen is prepared by interweaving monofilaments having an appropriatediameter to give the desired opening size. For example, themonofilaments can be made of a metal, e.g., stainless steel. As theopening sizes get smaller, structural demands on the monofilaments maybecome greater. For example, for opening sizes less than 0.40 mm, it canbe advantageous to make the screens from monofilaments made from amaterial other than stainless steel, e.g., titanium, titanium alloys,amorphous metals, nickel, tungsten, rhodium, rhenium, ceramics, orglass. In some embodiments, the screen is made from a plate, e.g. ametal plate, having apertures, e.g., cut into the plate using a laser.In some embodiments, the open area of the mesh is less than 52%, e.g.,less than 41%, less than 36%, less than 31%, less than 30%.

In some embodiments, the second fibrous is sheared and passed throughthe first screen, or a different sized screen. In some embodiments, thesecond fibrous material is passed through a second screen having anaverage opening size equal to or less than that of first screen.

Referring to FIG. 4, a third fibrous material 220 can be prepared fromthe second fibrous material 216 by shearing the second fibrous material216 and passing the resulting material through a second screen 222having an average opening size less than the first screen 214.

Generally, the fibers of the fibrous materials can have a relativelylarge average length-to-diameter ratio (e.g., greater than 20-to-1),even if they have been sheared more than once. In addition, the fibersof the fibrous materials described herein may have a relatively narrowlength and/or length-to-diameter ratio distribution.

As used herein, average fiber widths (i.e., diameters) are thosedetermined optically by randomly selecting approximately 5,000 fibers.Average fiber lengths are corrected length-weighted lengths. BET(Brunauer, Emmet and Teller) surface areas are multi-point surfaceareas, and porosities are those determined by mercury porosimetry.

The average length-to-diameter ratio of the second fibrous material 14can be, e.g. greater than 8/1, e.g., greater than 10/1, greater than15/1, greater than 20/1, greater than 25/1, or greater than 50/1. Anaverage length of the second fibrous material 14 can be, e.g., betweenabout 0.5 mm and 2.5 mm, e.g., between about 0.75 mm and 1.0 mm, and anaverage width (i.e., diameter) of the second fibrous material 14 can be,e.g., between about 5 μm and 50 μm, e.g., between about 10 μm and 30 μm.

In some embodiments, a standard deviation of the length of the secondfibrous material 14 is less than 60 percent of an average length of thesecond fibrous material 14, e.g., less than 50 percent of the averagelength, less than 40 percent of the average length, less than 25 percentof the average length, less than 10 percent of the average length, lessthan 5 percent of the average length, or even less than 1 percent of theaverage length.

In some embodiments, a BET surface area of the second fibrous materialis greater than 0.1 m²/g, e.g., greater than 0.25 m²/g, greater than 0.5m²/g, greater than 1.0 m²/g, greater than 1.5 m²/g, greater than 1.75m²/g, greater than 5.0 m²/g, greater than 10 m²/g, greater than 25 m²/g,greater than 35 m²/g, greater than 50 m²/g, greater than 60 m²/g,greater than 75 m²/g, greater than 100 m²/g, greater than 150 m²/g,greater than 200 m²/g, or even greater than 250 m²/g. A porosity of thesecond fibrous material 14 can be, e.g., greater than 20 percent,greater than 25 percent, greater than 35 percent, greater than 50percent, greater than 60 percent, greater than 70 percent, e.g., greaterthan 80 percent, greater than 85 percent, greater than 90 percent,greater than 92 percent, greater than 94 percent, greater than 95percent, greater than 97.5 percent, greater than 99 percent, or evengreater than 99.5 percent.

In some embodiments, a ratio of the average length-to-diameter ratio ofthe first fibrous material to the average length-to-diameter ratio ofthe second fibrous material is, e.g., less than 1.5, e.g., less than1.4, less than 1.25, less than 1.1, less than 1.075, less than 1.05,less than 1.025, or even substantially equal to 1.

In particular embodiments, the second fibrous material is sheared againand the resulting fibrous material passed through a second screen havingan average opening size less than the first screen to provide a thirdfibrous material. In such instances, a ratio of the averagelength-to-diameter ratio of the second fibrous material to the averagelength-to-diameter ratio of the third fibrous material can be, e.g.,less than 1.5, e.g., less than 1.4, less than 1.25, or even less than1.1.

In some embodiments, the third fibrous material is passed through athird screen to produce a fourth fibrous material. The fourth fibrousmaterial can be, e.g., passed through a fourth screen to produce a fifthmaterial. Similar screening processes can be repeated as many times asdesired to produce the desired fibrous material having the desiredproperties.

Densification

Densified materials can be processed by any of the methods describedherein, or any material described herein, e.g., any fibrous materialdescribed herein, can be processed by any one or more methods describedherein, and then densified as described herein.

A material, e.g., a fibrous material, having a low bulk density can bedensified to a product having a higher bulk density. For example, amaterial composition having a bulk density of 0.05 g/cm³ can bedensified by sealing the fibrous material in a relatively gasimpermeable structure, e.g., a bag made of polyethylene or a bag made ofalternating layers of polyethylene and a nylon, and then evacuating theentrapped gas, e.g., air, from the structure. After evacuation of theair from the structure, the fibrous material can have, e.g., a bulkdensity of greater than 0.3 g/cm³, e.g., 0.5 g/cm³, 0.6 g/cm³, 0.7 g/cm³or more, e.g., 0.85 g/cm³. After densification, the product canprocessed by any of the methods described herein, e.g., irradiated,e.g., with gamma radiation. This can be advantageous when it isdesirable to transport the material to another location, e.g., a remotemanufacturing plant, where the fibrous material composition can be addedto a solution, e.g., to produce ethanol. After piercing thesubstantially gas impermeable structure, the densified fibrous materialcan revert to nearly its initial bulk density, e.g., greater than 60percent of its initial bulk density, e.g., 70 percent, 80 percent, 85percent or more, e.g., 95 percent of its initial bulk density. To reducestatic electricity in the fibrous material, an anti-static agent can beadded to the material.

In some embodiments, the structure, e.g., bag, is formed of a materialthat dissolves in a liquid, such as water. For example, the structurecan be formed from a polyvinyl alcohol so that it dissolves when incontact with a water-based system. Such embodiments allow densifiedstructures to be added directly to solutions that include amicroorganism, without first releasing the contents of the structure,e.g., by cutting.

Referring to FIG. 5, a biomass material can be combined with any desiredadditives and a binder, and subsequently densified by application ofpressure, e.g., by passing the material through a nip defined betweencounter-rotating pressure rolls or by passing the material through apellet mill. During the application of pressure, heat can optionally beapplied to aid in the densification of the fibrous material. Thedensified material can then be irradiated.

In some embodiments, the material prior to densification has a bulkdensity of less than 0.25 g/cm³, e.g., 0.20 g/cm³, 0.15 g/cm³, 0.10g/cm³, 0.05 g/cm³ or less, e.g., 0.025 g/cm³. Bulk density is determinedusing ASTM D1895B. Briefly, the method involves filling a measuringcylinder of known volume with a sample and obtaining a weight of thesample. The bulk density is calculated by dividing the weight of thesample in grams by the known volume of the cylinder in cubiccentimeters.

The preferred binders include binders that are soluble in water, swollenby water, or that has a glass transition temperature of less 25° C., asdetermined by differential scanning calorimetry. By water-solublebinders, we mean binders having a solubility of at least about 0.05weight percent in water. By water swellable binders, we mean bindersthat increase in volume by more than 0.5 percent upon exposure to water.

In some embodiments, the binders that are soluble or swollen by waterinclude a functional group that is capable of forming a bond, e.g., ahydrogen bond, with the fibers of the fibrous material, e.g., cellulosicfibrous material. For example, the functional group can be a carboxylicacid group, a carboxylate group, a carbonyl group, e.g., of an aldehydeor a ketone, a sulfonic acid group, a sulfonate group, a phosphoric acidgroup, a phosphate group, an amide group, an amine group, a hydroxylgroup, e.g., of an alcohol, and combinations of these groups, e.g., acarboxylic acid group and a hydroxyl group. Specific monomeric examplesinclude glycerin, glyoxal, ascorbic acid, urea, glycine,pentaerythritol, a monosaccharide or a disaccharide, citric acid, andtartaric acid. Suitable saccharides include glucose, sucrose, lactose,ribose, fructose, mannose, arabinose and erythrose. Polymeric examplesinclude polyglycols, polyethylene oxide, polycarboxylic acids,polyamides, polyamines and polysulfonic acids polysulfonates. Specificpolymeric examples include polypropylene glycol (PPG), polyethyleneglycol (PEG), polyethylene oxide, e.g., POLYOX®, copolymers of ethyleneoxide and propylene oxide, polyacrylic acid (PAA), polyacrylamide,polypeptides, polyethylenimine, polyvinylpyridine,poly(sodium-4-styrenesulfonate) andpoly(2-acrylamido-methyl-1-propanesulfonic acid).

In some embodiments, the binder includes a polymer that has a glasstransition temperature less 25° C. Examples of such polymers includethermoplastic elastomers (TPEs). Examples of TPEs include polyetherblock amides, such as those available under the tradename PEBAX®,polyester elastomers, such as those available under the tradenameHYTREL®, and styrenic block copolymers, such as those available underthe tradename KRATON®. Other suitable polymers having a glass transitiontemperature less 25° C. include ethylene vinyl acetate copolymer (EVA),polyolefins, e.g., polyethylene, polypropylene, ethylene-propylenecopolymers, and copolymers of ethylene and alpha olefins, e.g.,1-octene, such as those available under the trade name ENGAGE®. In someembodiments, e.g., when the material is a fiberized polycoated paper,the material is densified without the addition of a separate low glasstransition temperature polymer.

In a particular embodiment, the binder is a lignin, e.g., a natural orsynthetically modified lignin.

A suitable amount of binder added to the material, calculated on a dryweight basis, is, e.g., from about 0.01 percent to about 50 percent,e.g., 0.03 percent, 0.05 percent, 0.1 percent, 0.25 percent, 0.5percent, 1.0 percent, 5 percent, 10 percent or more, e.g., 25 percent,based on a total weight of the densified material. The binder can beadded to the material as a neat, pure liquid, as a liquid having thebinder dissolved therein, as a dry powder of the binder, or as pelletsof the binder.

The densified fibrous material can be made in a pellet mill. Referringto FIG. 6, a pellet mill 300 has a hopper 301 for holding undensifiedmaterial 310 that includes a carbohydrate-containing materials, such ascellulose. The hopper communicates with an auger 312 that is driven byvariable speed motor 314 so that undensified material can be transportedto a conditioner 320 that stirs the undensified material with paddles322 that are rotated by conditioner motor 330. Other ingredients, e.g.,any of the additives and/or fillers described herein, can be added atinlet 332. If desired, heat may be added while the fibrous material isin conditioner. After conditioned, the material passes from theconditioner through a dump chute 340, and to another auger 342. The dumpchute, as controlled by actuator 344, allows for unobstructed passage ofthe material from conditioner to auger. Auger is rotated by motor 346,and controls the feeding of the fibrous material into die and rollerassembly 350. Specifically, the material is introduced into a hollow,cylindrical die 352, which rotates about a horizontal axis and which hasradially extending die holes 250. Die 352 is rotated about the axis bymotor 360, which includes a horsepower gauge, indicating total powerconsumed by the motor. Densified material 370, e.g., in the form ofpellets, drops from chute 372 and are captured and processed, such as byirradiation.

The material, after densification, can be conveniently in the form ofpellets or chips having a variety of shapes. The pellets can then beirradiated. In some embodiments, the pellets or chips are cylindrical inshape, e.g., having a maximum transverse dimension of, e.g., 1 mm ormore, e.g., 2 mm, 3 mm, 5 mm, 8 mm, 10 mm, 15 mm or more, e.g., 25 mm.Another convenient shape for making composites includes pellets or chipsthat are plate-like in form, e.g., having a thickness of 1 mm or more,e.g., 2 mm, 3 mm, 5 mm, 8 mm, 10 mm or more, e.g., 25 mm; a width of,e.g., 5 mm or more, e.g., 10 mm, 15 mm, 25 mm, 30 mm or more, e.g., 50mm; and a length of 5 mm or more, e.g., 10 mm, 15 mm, 25 mm, 30 mm ormore, e.g., 50 mm.

Referring now FIG. 7A-7D, pellets can be made so that they have a hollowinside. As shown, the hollow can be generally in-line with the center ofthe pellet (FIG. 7B), or out of line with the center of the pellet (FIG.7C). Making the pellet hollow inside can increase the rate ofdissolution in a liquid after irradiation.

Referring now to FIG. 7D, the pellet can have, e.g., a transverse shapethat is multi-lobal, e.g., tri-lobal as shown, or tetra-lobal,penta-lobal, hexa-lobal or deca-lobal. Making the pellets in suchtransverse shapes can also increase the rate of dissolution in asolution after irradiation.

Alternatively, the densified material can be in any other desired form,e.g., the densified material can be in the form of a mat, roll or bale.

EXAMPLES

In one example, half-gallon juice cartons made of un-printed white Kraftboard having a bulk density of 20 lb/ft³ can be used as a feedstock.Cartons can be folded flat and then fed into a shredder to produce aconfetti-like material having a width of between 0.1 inch and 0.5 inch,a length of between 0.25 inch and 1 inch and a thickness equivalent tothat of the starting material (about 0.075 inch). The confetti-likematerial can be fed to a rotary knife cutter, which shears theconfetti-like pieces, tearing the pieces apart and releasing fibrousmaterial.

In some cases, multiple shredder-shearer trains can be arranged inseries with output. In one embodiment, two shredder-shearer trains canbe arranged in series with output from the first shearer fed as input tothe second shredder. In another embodiment, three shredder-shearertrains can be arranged in series with output from the first shearer fedas input to the second shredder and output from the second shearer fedas input to the third shredder. Multiple passes through shredder-shearertrains are anticipated to increase decrease particle size and increaseoverall surface area within the feedstream.

In another example, fibrous material produced from shredding andshearing juice cartons can be treated to increase its bulk density. Insome cases, the fibrous material can be sprayed with water or a dilutestock solution of POLYOX™ WSR N10 (polyethylene oxide) prepared inwater. The wetted fibrous material can then be processed through apellet mill operating at room temperature. The pellet mill can increasethe bulk density of the feedstream by more than an order of magnitude.

Pretreatment

Physically prepared feedstock can be pretreated for use in primaryproduction processes by, for example, reducing the average molecularweight and crystallinity of the feedstock and/or increasing the surfacearea and/or porosity of the feedstock. Pretreatment processes caninclude one or more of irradiation, sonication, oxidation, pyrolysis,and steam explosion. The various pretreatment systems and methods can beused in combinations of two, three, or even four of these technologies.

Pretreatment Combinations

In some embodiments, biomass can be processed by applying two or more ofany of the processes described herein, such as two or more of radiation,sonication, oxidation, pyrolysis, and steam explosion either with orwithout prior, intermediate, or subsequent feedstock preparation asdescribed herein. The processes can be applied in any order (orconcurrently) to the biomass, e.g., a cellulosic and/or lignocellulosicmaterial and/or a starchy material, such as kernels of corn. In otherembodiments, materials that include a carbohydrate are prepared byapplying three, four or more of any of the processes described herein(in any order or concurrently). For example, a carbohydrate can beprepared by applying radiation, sonication, oxidation, pyrolysis, and,optionally, steam explosion to a cellulosic and/or lignocellulosicmaterial (in any order or concurrently). The providedcarbohydrate-containing material can then be converted by one or moremicroorganisms, such as bacteria, yeast, or mixtures of yeast andbacteria, to a number of desirable products, as described herein.Multiple processes can provide materials that can be more readilyutilized by a variety of microorganisms because of their lower molecularweight, lower crystallinity, and/or enhanced solubility. Multipleprocesses can provide synergies and can reduce overall energy inputrequired in comparison to any single process.

For example, in some embodiments, feedstocks are provided that include acarbohydrate that is produced by a process that includes irradiating andsonicating (in either order or concurrently) a cellulosic and/or alignocellulosic material, a process that includes irradiating andoxidizing (in either order or concurrently) a cellulosic and/or alignocellulosic material, a process that includes irradiating andpyrolyzing (in either order or concurrently) a cellulosic and/or alignocellulosic material, a process that includes irradiating andpyrolyzing (in either order or concurrently) a cellulosic and/or alignocellulosic material, or a process that includes irradiating andsteam-exploding (in either order or concurrently) a cellulosic and/or alignocellulosic material. The provided feedstock can then be contactedwith a microorganism having the ability to convert at least a portion,e.g., at least about 1 percent by weight, of the feedstock to theproduct, such as the combustible fuel, as described herein.

In some embodiments, the process does not include hydrolyzing thecellulosic and/or lignocellulosic material, such as with an acid or abase, e.g., a mineral acid, such as hydrochloric or sulfuric acid.

If desired, some or none of the feedstock can include a hydrolyzedmaterial. For example, in some embodiments, at least about seventypercent by weight of the feedstock is an unhydrolyzed material, e.g., atleast at 95 percent by weight of the feedstock is an unhydrolyzedmaterial. In some embodiments, substantially all of the feedstock is anunhydrolyzed material.

Any feedstock or any reactor or fermentor charged with a feedstock caninclude a buffer, such as sodium bicarbonate, ammonium chloride or Tris;an electrolyte, such as potassium chloride, sodium chloride, or calciumchloride; a growth factor, such as biotin and/or a base pair such asuracil or an equivalent thereof; a surfactant, such as Tween® orpolyethylene glycol; a mineral, such as such as calcium, chromium,copper, iodine, iron, selenium, or zinc; or a chelating agent, such asethylene diamine, ethylene diamine tetraacetic acid (EDTA) (or its saltform, e.g., sodium or potassium EDTA), or dimercaprol.

When radiation is utilized, it can be applied to any sample that is dryor wet, or even dispersed in a liquid, such as water. For example,irradiation can be performed on cellulosic and/or lignocellulosicmaterial in which less than about 25 percent by weight of the cellulosicand/or lignocellulosic material has surfaces wetted with a liquid, suchas water. In some embodiments, irradiating is performed on cellulosicand/or lignocellulosic material in which substantially none of thecellulosic and/or lignocellulosic material is wetted with a liquid, suchas water.

In some embodiments, any processing described herein occurs after thecellulosic and/or lignocellulosic material remains dry as acquired orhas been dried, e.g., using heat and/or reduced pressure. For example,in some embodiments, the cellulosic and/or lignocellulosic material hasless than about five percent by weight retained water, measured at 25°C. and at fifty percent relative humidity.

If desired, a swelling agent, as defined herein, can be utilized in anyprocess described herein. In some embodiments, when a cellulosic and/orlignocellulosic material is processed using radiation, less than about25 percent by weight of the cellulosic and/or lignocellulosic materialis in a swollen state, the swollen state being characterized as having avolume of more than about 2.5 percent higher than an unswollen state,e.g., more than 5.0, 7.5, 10, or 15 percent higher than the unswollenstate. In some embodiments, when radiation is utilized on a cellulosicand/or lignocellulosic material, substantially none of the cellulosicand/or lignocellulosic material is in a swollen state.

In specific embodiments when radiation is utilized, the cellulosicand/or lignocellulosic material includes a swelling agent, and swollencellulosic and/or lignocellulosic receives a dose of less than about 10Mrad.

When radiation is utilized in any process, it can be applied while thecellulosic and/or lignocellulosic is exposed to air, oxygen-enrichedair, or even oxygen itself, or blanketed by an inert gas such asnitrogen, argon, or helium. When maximum oxidation is desired, anoxidizing environment is utilized, such as air or oxygen and thedistance from the radiation source is optimized to maximize reactive gasformation, e.g., ozone and/or oxides of nitrogen.

When radiation is utilized, it may be applied to biomass, such ascellulosic and/or lignocellulosic material, under a pressure of greaterthan about 2.5 atmospheres, such as greater than 5, 10, 15, 20 or evengreater than about 50 atmospheres.

In specific embodiments, the process includes irradiating and sonicatingand irradiating precedes sonicating. In other specific embodiments,sonication precedes irradiating, or irradiating and sonicating occurconcurrently.

In some embodiments, the process includes irradiating and sonicating (ineither order or concurrently) and further includes oxidizing, pyrolyzingor steam exploding.

When the process includes radiation, the irradiating can be performedutilizing an ionizing radiation, such as gamma rays, x-rays, energeticultraviolet radiation, such as ultraviolet C radiation having awavelength of from about 100 nm to about 280 nm, a beam of particles,such as a beam of electrons, slow neutrons or alpha particles. In someembodiments, irradiating includes two or more radiation sources, such asgamma rays and a beam of electrons, which can be applied in either orderor concurrently.

In specific embodiments, sonicating can performed at a frequency ofbetween about 15 kHz and about 25 kHz, such as between about 18 kHz and22 kHz utilizing a 1 KW or larger horn, e.g., a 2, 3, 4, 5, or even a 10KW horn.

In some embodiments, the cellulosic and/or lignocellulosic materialincludes a first cellulose having a first number average molecularweight and the resulting carbohydrate includes a second cellulose havinga second number average molecular weight lower than the first numberaverage molecular weight. For example, the second number averagemolecular weight is lower than the first number average molecular weightby more than about twenty-five percent, e.g., 2×, 3×, 5×, 7×, 10×, 25×,even 100× reduction.

In some embodiments, the first cellulose has a first crystallinity andthe second cellulose has a second crystallinity lower than the firstcrystallinity, such as lower than about two, three, five, ten, fifteenor twenty-five percent lower.

In some embodiments, the first cellulose has a first level of oxidationand the second cellulose has a second level of oxidation higher than thefirst level of oxidation, such as two, three, four, five, ten or eventwenty-five percent higher.

In one example of the use of radiation with oxidation as a pretreatment,half-gallon juice cartons made of un-printed polycoated white Kraftboard having a bulk density of 20 lb/ft³ are used as a feedstock.Cartons are folded flat and then fed into a sequence of threeshredder-shearer trains arranged in series with output from the firstshearer fed as input to the second shredder, and output from the secondshearer fed as input to the third shredder. The fibrous materialproduced by the can be sprayed with water and processed through a pelletmill operating at room temperature. The densified pellets can be placedin a glass ampoule, which is sealed under an atmosphere of air. Thepellets in the ampoule are irradiated with gamma radiation for about 3hours at a dose rate of about 1 Mrad per hour to provide an irradiatedmaterial in which the cellulose has a lower molecular weight than thefibrous Kraft starting material.

Radiation Treatment

One or more irradiation processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences. Irradiation can reduce the molecular weight and/orcrystallinity of feedstock. In some embodiments, energy deposited in amaterial that releases an electron from its atomic orbital is used toirradiate the materials. The radiation may be provided by 1) heavycharged particles, such as alpha particles or protons, 2) electrons,produced, for example, in beta decay or electron beam accelerators, or3) electromagnetic radiation, for example, gamma rays, x rays, orultraviolet rays. In one approach, radiation produced by radioactivesubstances can be used to irradiate the feedstock. In some embodiments,any combination in any order or concurrently of (1) through (3) may beutilized. In another approach, electromagnetic radiation (e.g., producedusing electron beam emitters) can be used to irradiate the feedstock.The doses applied depend on the desired effect and the particularfeedstock. For example, high doses of radiation can break chemical bondswithin feedstock components and low doses of radiation can increasechemical bonding (e.g., cross-linking) within feedstock components. Insome instances when chain scission is desirable and/or polymer chainfunctionalization is desirable, particles heavier than electrons, suchas protons, helium nuclei, argon ions, silicon ions, neon ions, carbonions, phosphorus ions, oxygen ions or nitrogen ions can be utilized.When ring-opening chain scission is desired, positively chargedparticles can be utilized for their Lewis acid properties for enhancedring-opening chain scission. For example, when oxygen-containingfunctional groups are desired, irradiation in the presence of oxygen oreven irradiation with oxygen ions can be performed. For example, whennitrogen-containing functional groups are desirable, irradiation in thepresence of nitrogen or even irradiation with nitrogen ions can beperformed.

Referring to FIG. 8, in one method, a first material 2 that is orincludes cellulose having a first number average molecular weight(^(T)M_(N1)) is irradiated, e.g., by treatment with ionizing radiation(e.g., in the form of gamma radiation, X-ray radiation, 100 nm to 280 nmultraviolet (UV) light, a beam of electrons or other charged particles)to provide a second material 3 that includes cellulose having a secondnumber average molecular weight (^(T)M_(N2)) lower than the first numberaverage molecular weight. The second material (or the first and secondmaterial) can be combined with a microorganism (e.g., a bacterium or ayeast) that can utilize the second and/or first material to produce afuel 5 that is or includes hydrogen, an alcohol (e.g., ethanol orbutanol, such as n-, sec- or t-butanol), an organic acid, a hydrocarbonor mixtures of any of these.

Since the second material 3 has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble in a solution containing amicroorganism. These properties make the second material 3 moresusceptible to chemical, enzymatic and/or biological attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Radiationcan also sterilize the materials.

In some embodiments, the second number average molecular weight (M_(N2))is lower than the first number average molecular weight (^(T)M_(N1)) bymore than about 10 percent, e.g., 15, 20, 25, 30, 35, 40, 50 percent, 60percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has ascrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior toirradiation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after irradiation is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in some embodiments, e.g., after extensiveirradiation, it is possible to have a crystallinity index of lower than5 percent. In some embodiments, the material after irradiation issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto irradiation) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after irradiation is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive irradiation, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or biologicalattack. In some embodiments, to increase the level of the oxidation ofthe second material relative to the first material, the irradiation isperformed under an oxidizing environment, e.g., under a blanket of airor oxygen, producing a second material that is more oxidized than thefirst material. For example, the second material can have more hydroxylgroups, aldehyde groups, ketone groups, ester groups or carboxylic acidgroups, which can increase its hydrophilicity.

Ionizing Radiation

Each form of radiation ionizes the biomass via particular interactions,as determined by the energy of the radiation. Heavy charged particlesprimarily ionize matter via Coulomb scattering; furthermore, theseinteractions produce energetic electrons that may further ionize matter.Alpha particles are identical to the nucleus of a helium atom and areproduced by the alpha decay of various radioactive nuclei, such asisotopes of bismuth, polonium, astatine, radon, francium, radium,several actinides, such as actinium, thorium, uranium, neptunium,curium, californium, americium, and plutonium.

When particles are utilized, they can be neutral (uncharged), positivelycharged or negatively charged. When charged, the charged particles canbear a single positive or negative charge, or multiple charges, e.g.,one, two, three or even four or more charges. In instances in whichchain scission is desired, positively charged particles may bedesirable, in part, due to their acidic nature. When particles areutilized, the particles can have the mass of a resting electron, orgreater, e.g., 500, 1000, 1500, or 2000 or more, e.g., 10,000 or even100,000 times the mass of a resting electron. For example, the particlescan have a mass of from about 1 atomic unit to about 150 atomic units,e.g., from about 1 atomic unit to about 50 atomic units, or from about 1to about 25, e.g., 1, 2, 3, 4, 5, 10, 12 or 15 amu. Accelerators used toaccelerate the particles can be electrostatic DC, electrodynamic DC, RFlinear, magnetic induction linear or continuous wave. For example,cyclotron type accelerators are available from IBA, Belgium, such as theRhodotron® system, while DC type accelerators are available from RDI,now IBA Industrial, such as the Dynamitron®. Exemplary ions and ionaccelerators are discussed in Introductory Nuclear Physics, Kenneth S.Krane, John Wiley & Sons, Inc. (1988), Krsto Prelec, FIZIKA B 6 (1997)4, 177-206, Chu, William T., “Overview of Light-Ion Beam Therapy”,Columbus-Ohio, ICRU-IAEA Meeting, 18-20 Mar. 2006, Iwata, Y. et al.,“Alternating-Phase-Focused IH-DTL for Heavy-Ion Medical Accelerators”,Proceedings of EPAC 2006, Edinburgh, Scotland, and Leitner, C. M. etal., “Status of the Superconducting ECR Ion Source Venus”, Proceedingsof EPAC 2000, Vienna, Austria.

Electrons interact via Coulomb scattering and bremsstrahlung radiationproduced by changes in the velocity of electrons. Electrons may beproduced by radioactive nuclei that undergo beta decay, such as isotopesof iodine, cesium, technetium, and iridium. Alternatively, an electrongun can be used as an electron source via thermionic emission.

Electromagnetic radiation interacts via three processes: photoelectricabsorption, Compton scattering, and pair production. The dominatinginteraction is determined by the energy of the incident radiation andthe atomic number of the material. The summation of interactionscontributing to the absorbed radiation in cellulosic material can beexpressed by the mass absorption coefficient (see “Ionization Radiation”in PCT/US2007/022719).

Electromagnetic radiation can be subclassified as gamma rays, x rays,ultraviolet rays, infrared rays, microwaves, or radio waves, dependingon wavelength.

For example, gamma radiation can be employed to irradiate the materials.Referring to FIGS. 9 and 10 (an enlarged view of region R), a gammairradiator 10 includes gamma radiation sources 408, e.g., ⁶⁰Co pellets,a working table 14 for holding the materials to be irradiated andstorage 16, e.g., made of a plurality iron plates, all of which arehoused in a concrete containment chamber (vault) 20 that includes a mazeentranceway 22 beyond a lead-lined door 26. Storage 16 includes aplurality of channels 30, e.g., sixteen or more channels, allowing thegamma radiation sources to pass through storage on their way proximatethe working table.

In operation, the sample to be irradiated is placed on a working table.The irradiator is configured to deliver the desired dose rate andmonitoring equipment is connected to an experimental block 31. Theoperator then leaves the containment chamber, passing through the mazeentranceway and through the lead-lined door. The operator mans a controlpanel 32, instructing a computer 33 to lift the radiation sources 12into working position using cylinder 36 attached to a hydraulic pump 40.

Gamma radiation has the advantage of a significant penetration depthinto a variety of material in the sample. Sources of gamma rays includeradioactive nuclei, such as isotopes of cobalt, calcium, technicium,chromium, gallium, indium, iodine, iron, krypton, samarium, selenium,sodium, thalium, and xenon.

Sources of x rays include electron beam collision with metal targets,such as tungsten or molybdenum or alloys, or compact light sources, suchas those produced commercially by Lyncean.

Sources for ultraviolet radiation include deuterium or cadmium lamps.

Sources for infrared radiation include sapphire, zinc, or selenidewindow ceramic lamps.

Sources for microwaves include klystrons, Slevin type RF sources, oratom beam sources that employ hydrogen, oxygen, or nitrogen gases.

Electron Beam

In some embodiments, a beam of electrons is used as the radiationsource. A beam of electrons has the advantages of high dose rates (e.g.,1, 5, or even 10 Mrad per second), high throughput, less containment,and less confinement equipment. Electrons can also be more efficient atcausing chain scission. In addition, electrons having energies of 4-10MeV can have a penetration depth of 5 to 30 mm or more, such as 40 mm.

Electron beams can be generated, e.g., by electrostatic generators,cascade generators, transformer generators, low energy accelerators witha scanning system, low energy accelerators with a linear cathode, linearaccelerators, and pulsed accelerators. Electrons as an ionizingradiation source can be useful, e.g., for relatively thin piles ofmaterials, e.g., less than 0.5 inch, e.g., less than 0.4 inch, 0.3 inch,0.2 inch, or less than 0.1 inch. In some embodiments, the energy of eachelectron of the electron beam is from about 0.3 MeV to about 2.0 MeV(million electron volts), e.g., from about 0.5 MeV to about 1.5 MeV, orfrom about 0.7 MeV to about 1.25 MeV.

In some embodiments, electrons used to treat biomass material can haveaverage energies of 0.05 c or more (e.g., 0.10 c or more, 0.2 c or more,0.3 c or more, 0.4 c or more, 0.5 c or more, 0.6 c or more, 0.7 c ormore, 0.8 c or more, 0.9 c or more, 0.99 c or more, 0.9999 c or more),where c corresponds to the vacuum velocity of light.

FIG. 11A shows a process flow diagram 3000 that includes various stepsin an electron beam irradiation feedstock pretreatment sequence. Infirst step 3010, a supply of dry feedstock is received from a feedsource. As discussed above, the dry feedstock from the feed source maybe pre-processed prior to delivery to the electron beam irradiationdevices. For example, if the feedstock is derived from plant sources,certain portions of the plant material may be removed prior tocollection of the plant material and/or before the plant material isdelivered by the feedstock transport device. Alternatively, or inaddition, as expressed in optional step 3020, the biomass feedstock canbe subjected to mechanical processing (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the electronbeam irradiation devices.

In step 3030, the dry feedstock is transferred to a feedstock transportdevice (e.g., a conveyor belt) and is distributed over thecross-sectional width of the feedstock transport device approximatelyuniformly by volume. This can be accomplished, for example, manually orby inducing a localized vibration motion at some point in the feedstocktransport device prior to the electron beam irradiation processing.

In some embodiments, a mixing system introduces a chemical agent 3045into the feedstock in an optional process 3040 that produces a slurry.Combining water with the processed feedstock in mixing step 3040 createsan aqueous feedstock slurry that may be transported through, forexample, piping rather than using, for example, a conveyor belt.

The next step 3050 is a loop that encompasses exposing the feedstock (indry or slurry form) to electron beam radiation via one or more (say, N)electron beam irradiation devices. The feedstock slurry is moved througheach of the N “showers” of electron beams at step 3052. The movement mayeither be at a continuous speed through and between the showers, orthere may be a pause through each shower, followed by a sudden movementto the next shower. A small slice of the feedstock slurry is exposed toeach shower for some predetermined exposure time at step 3053.

Electron beam irradiation devices may be procured commercially from IonBeam Applications, Louvain-la-Neuve, Belgium or the Titan Corporation,San Diego, Calif. Typical electron energies can be 1 MeV, 2 MeV, 4.5MeV, 7.5 MeV, or 10 MeV. Typical electron beam irradiation device powercan be 1 kW, 5 kW, 10 kW, 20 kW, 50 kW, 100 kW, 250 kW, or 500 kW.Effectiveness of depolymerization of the feedstock slurry depends on theelectron energy used and the dose applied, while exposure time dependson the power and dose. Typical doses may take values of 1 kGy, 5 kGy, 10kGy, 20 kGy, 50 kGy, 100 kGy, or 200 kGy.

Tradeoffs in considering electron beam irradiation device powerspecifications include cost to operate, capital costs, depreciation, anddevice footprint. Tradeoffs in considering exposure dose levels ofelectron beam irradiation would be energy costs and environment, safety,and health (ESH) concerns. Typically, irradiation devices are housed ina vault, e.g., of lead or concrete.

Tradeoffs in considering electron energies include energy costs; a lowerelectron energy may be advantageous in encouraging depolymerization ofcertain feedstock slurry (see, for example, Bouchard, et al, Cellulose(2006) 13: 601-610).

It may be advantageous to provide a double-pass of electron beamirradiation in order to provide a more effective depolymerizationprocess. For example, the feedstock transport device could direct thefeedstock (in dry or slurry form) underneath and in a reverse directionto its initial transport direction. Double-pass systems can allowthicker feedstock slurries to be processed and can provide a moreuniform depolymerization through the thickness of the feedstock slurry.

The electron beam irradiation device can produce either a fixed beam ora scanning beam. A scanning beam may be advantageous with large scansweep length and high scan speeds, as this would effectively replace alarge, fixed beam width. Further, available sweep widths of 0.5 m, 1 m,2 m or more are available.

Once a portion of feedstock slurry has been transported through the Nelectron beam irradiation devices, it may be necessary in someembodiments, as in step 3060, to mechanically separate the liquid andsolid components of the feedstock slurry. In these embodiments, a liquidportion of the feedstock slurry is filtered for residual solid particlesand recycled back to the slurry preparation step 3040. A solid portionof the feedstock slurry is then advanced on to the next processing step3070 via the feedstock transport device. In other embodiments, thefeedstock is maintained in slurry form for further processing.

Ion Particle Beams

Particles heavier than electrons can be utilized to irradiatecarbohydrates or materials that include carbohydrates, e.g., cellulosicmaterials, lignocellulosic materials, starchy materials, or mixtures ofany of these and others described herein. For example, protons, heliumnuclei, argon ions, silicon ions, neon ions carbon ions, phosphorusions, oxygen ions or nitrogen ions can be utilized. In some embodiments,particles heavier than electrons can induce higher amounts of chainscission. In some instances, positively charged particles can inducehigher amounts of chain scission than negatively charged particles dueto their acidity.

Heavier particle beams can be generated, e.g., using linear acceleratorsor cyclotrons. In some embodiments, the energy of each particle of thebeam is from about 1.0 MeV/atomic unit to about 6,000 MeV/atomic unit,e.g., from about 3 MeV/atomic unit to about 4,800 MeV/atomic unit, orfrom about 10 MeV/atomic unit to about 1,000 MeV/atomic unit.

The types and properties of particles that can be used to irradiatevarious types of biomass materials are disclosed in further detailbelow. Further, systems and methods for producing beams of suchparticles are disclosed.

1. Types of Ions

In general, many different types of ions can be used to irradiatebiomass materials. For example, in some embodiments, ion beams caninclude relatively light ions, such as protons and/or helium ions. Incertain embodiments, the ion beams can include moderately heavier ions,such as carbon ions, nitrogen ions, oxygen ions, and/or neon ions. Insome embodiments, ion beams can include still heavier ions, such asargon ions, silicon ions, phosphorus ions, sodium ions, calcium ions,and/or iron ions.

In certain embodiments, ion beams used to irradiate biomass materialscan include more than one different type of ions. For example, ion beamscan include mixtures of two or more (e.g., three, or four or more)different types of ions. Exemplary mixtures can include carbon ions andprotons, carbon ions and oxygen ions, nitrogen ions and protons, andiron ions and protons. More generally, mixtures of any of the ionsdiscussed above (or any other ions) can be used to form irradiating ionbeams. In particular, mixtures of relatively light and relativelyheavier ions can be used in a single ion beam, where each of thedifferent types of ions has different effectiveness in irradiatingdifferent types of biomass materials.

In some embodiments, ion beams for irradiating biomass materials includepositively-charged ions. The positively charged ions can include, forexample, positively charged hydrogen ions (e.g., protons), noble gasions (e.g., helium, neon, argon), carbon ions, nitrogen ions, oxygenions, silicon atoms, phosphorus ions, and metal ions such as sodiumions, calcium ions, and/or iron ions. Without wishing to be bound by anytheory, it is believed that such positively-charged ions behavechemically as Lewis acid moieties when exposed to biomass materials,initiating and sustaining cationic ring-opening chain scission reactionsin an oxidative environment.

In certain embodiments, ion beams for irradiating biomass materialsinclude negatively-charged ions. Negatively charged ions can include,for example, negatively charged hydrogen ions (e.g., hydride ions), andnegatively charged ions of various relatively electronegative nuclei(e.g., oxygen ions, nitrogen ions, carbon ions, silicon ions, andphosphorus ions). Without wishing to be bound by any theory, it isbelieved that such negatively-charged ions behave chemically as Lewisbase moieties when exposed to biomass materials, causing anionicring-opening chain scission reactions in a reducing environment.

In some embodiments, beams for irradiating biomass materials can includeneutral atoms. For example, any one or more of hydrogen atoms, heliumatoms, carbon atoms, nitrogen atoms, oxygen atoms, neon atoms, siliconatoms, phosphorus atoms, argon atoms, and iron atoms can be included inbeams that are used for irradiation of biomass materials. In general,mixtures of any two or more of the above types of atoms (e.g., three ormore, four or more, or even more) can be present in the beams.

The preceding discussion has focused on ion beams that includemononuclear ions and/or neutral particles (e.g., atomic ions and neutralatoms). Typically, such particles are the easiest—in energetic terms—togenerate, and parent particles from which these species are generatedmay be available in abundant supply. However, in some embodiments, beamsfor irradiating biomass materials can include one or more types of ionsor neutral particles that are polynuclear, e.g., including two or moredifferent types of nuclei. For example, ion beams can include positiveand/or negative ions and/or neutral particles formed from species suchas N₂, O₂, H₂, CH₄, and other molecular species. Ion beams can alsoinclude ions and/or neutral particles formed from heavier species thatinclude even more nuclei, such as various hydrocarbon-based speciesand/or various inorganic species, including coordination compounds ofvarious metals.

In certain embodiments, ion beams used to irradiate biomass materialsinclude singly-charged ions such as one or more of H⁺, H⁻, He⁺, Ne⁺,Ar⁺, C⁺, C⁺, O⁺, O⁻, N⁺, N⁻, Si⁺, Si⁻, P⁺, P⁻, Na⁺, Ca⁺, and Fe⁺. Insome embodiments, ion beams can include multiply-charged ions such asone or more of C²⁺, C³⁺, C⁴⁺, N³⁺, N⁵⁺, N³⁻, O²⁺, O²⁻, O₂ ²⁻, Si²⁺,Si⁴⁺, Si²⁻, and Si⁴⁻. In general, the ion beams can also include morecomplex polynuclear ions that bear multiple positive or negativecharges. In certain embodiments, by virtue of the structure of thepolynuclear ion, the positive or negative charges can be effectivelydistributed over substantially the entire structure of the ions. In someembodiments, the positive or negative charges can be somewhat localizedover portions of the structure of the ions, by virtue of the electronicstructures of the ions.

2. Ion Generation

In this section, various methods for the generation of ions suitable forion beams are discussed. After the ions have been generated, they aretypically accelerated in one or more of various types of accelerators,and then directed to impinge on biomass materials. Accelerators and thestructures thereof will be discussed in more detail in the next section.

(i) Hydrogen Ions

Hydrogen ions can be generated using a variety of different methods inan ion source. Typically, hydrogen ions are introduced into an ionizingchamber of an ion source, and ions are produced by supplying energy togas molecules. During operation, such chambers can produce large ioncurrents suitable for seeding a downstream ion accelerator.

In some embodiments, hydrogen ions are produced via field ionization ofhydrogen gas. A schematic diagram of a field ionization source is shownin FIG. 43. Field ionization source 1100 includes a chamber 1170 whereionization of gas molecules (e.g., hydrogen gas molecules) occurs. Gasmolecules 1150 enter chamber 1170 by flowing along direction 1155 insupply tube 1120. Field ionization source 1100 includes an ionizationelectrode 1110. During operation, a large potential V_(E) (relative to acommon system ground potential) is applied to electrode 1110. Molecules1150 that circulate within a region adjacent to electrode 1110 areionized by the electric field that results from potential V_(E). Alsoduring operation, an extraction potential V_(X) is applied to extractors1130. The newly-formed ions migrate towards extractors 1130 under theinfluence of the electric fields of potentials V_(E) and V_(X). Ineffect, the newly-formed ions experience repulsive forces relative toionization electrode 1110, and attractive forces relative to extractors1130. As a result, certain of the newly-formed ions enter discharge tube1140, and propagate along direction 1165 under the influence ofpotentials V_(E) and V_(X).

Depending upon the sign of potential V_(E) (relative to the commonground potential), both positively and negatively charged ions can beformed. For example, in some embodiments, a positive potential can beapplied to electrode 1110 and a negative potential can be applied toextractors 1130. Positively charged hydrogen ions (e.g., protons H′)that are generated in chamber 1170 are repelled away from electrode 1110and toward extractors 1130. As a result, discharged particle stream 1160includes positively charged hydrogen ions that are transported to aninjector system.

In certain embodiments, a negative potential can be applied to electrode1110 and a positive potential can be applied to extractors 1130.Negatively charged hydrogen ions (e.g., hydride ions H⁻) that aregenerated in chamber 1170 are repelled away from electrode 1110 andtoward extractors 1130. Discharged particle stream 1160 includesnegatively charged hydrogen ions, which are then transported to aninjector system.

In some embodiments, both positive and negative hydrogen ions can beproduced via direct thermal heating of hydrogen gas. For example,hydrogen gas can be directed to enter a heating chamber that isevacuated to remove residual oxygen and other gases.

The hydrogen gas can then be heated via a heating element to produceionic species. Suitable heating elements include, for example, arcdischarge electrodes, heating filaments, heating coils, and a variety ofother thermal transfer elements.

In certain embodiments, when hydrogen ions are produced via either fieldemission or thermal heating, various hydrogen ion species can beproduced, including both positively and negatively charged ion species,and singly- and multiply-charged ion species. The various ion speciescan be separated from one another via one or more electrostatic and/ormagnetic separators. FIG. 44 shows a schematic diagram of anelectrostatic separator 1175 that is configured to separate a pluralityof hydrogen ion species from one another. Electrostatic separator 1175includes a pair of parallel electrodes 1180 to which a potential V_(S)is applied by a voltage source (not shown). Particle stream 1160,propagating in the direction indicated by the arrow, includes a varietyof positively- and negatively-charged, and singly- and multiply-charged,ion species. As the various ion species pass through electrodes 1180,the electric field between the electrodes deflects the ion trajectoriesaccording to the magnitude and sign of the ion species. In FIG. 44, forexample, the electric field points from the lower electrode toward theupper electrode in the region between electrodes 1180. As a result,positively-charged ions are deflected along an upward trajectory in FIG.44, and negatively-charged ions are deflected along a downwardtrajectory. Ion beams 1162 and 1164 each correspond topositively-charged ion species, with the ion species in ion beam 1162having a larger positive charge than the ion species is beam 1164 (e.g.,due to the larger positive charge of the ions in beam 1162, the beam isdeflected to a greater extent).

Similarly, ion beams 1166 and 1168 each correspond to negatively-chargedion species, with the ion species in ion beam 1168 having a largernegative charge than the ion species in ion beam 1166 (and thereby beingdeflected to a larger extent by the electric field between electrodes1180). Beam 1169 includes neutral particles originally present inparticle stream 1160; the neutral particles are largely unaffected bythe electric field between electrodes 1180, and therefore passundeflected through the electrodes. Each of the separated particlestreams enters one of delivery tubes 1192, 1194, 1196, 1198, and 1199,and can be delivered to an injector system for subsequent accelerationof the particles, or steered to be incident directly on the biomassmaterial. Alternatively, or in addition, any or all of the separatedparticle streams can be blocked to prevent ion and/or atomic speciesfrom reaching biomass material. As yet another alternative, certainparticle streams can be combined and then directed to an injector systemand/or steered to be incident directly on the biomass material usingknown techniques.

In general, particle beam separators can also use magnetic fields inaddition to, or rather than, electric fields for deflecting chargedparticles. In some embodiments, particle beam separators includemultiple pairs of electrodes, where each pair of electrodes generates anelectric field that deflects particles passing therethrough.Alternatively, or in addition, particle beam separators can include oneor more magnetic deflectors that are configured to deflect chargedparticles according to magnitude and sign of the particle charges.

(ii) Noble Gas Ions

Noble gas atoms (e.g., helium atoms, neon atoms, argon atoms) formpositively-charged ions when acted upon by relatively strong electricfields. Methods for generating noble gas ions therefore typicallyinclude generating a high-intensity electric field, and then introducingnoble gas atoms into the field region to cause field ionization of thegas atoms. A schematic diagram of a field ionization generator for noblegas ions (and also for other types of ions) is shown in FIG. 45. Fieldionization generator 1200 includes a tapered electrode 1220 positionedwithin a chamber 1210. A vacuum pump 1250 is in fluid communication withthe interior of chamber 1210 via inlet 1240, and reduces the pressure ofbackground gases within chamber 1210 during operation. One or more noblegas atoms 1280 are admitted to chamber 1210 via inlet tube 1230.

During operation, a relatively high positive potential V_(T) (e.g.,positive relative to a common external ground) is applied to taperedelectrode 1220. Noble gas atoms 1280 that enter a region of spacesurrounding the tip of electrode 1220 are ionized by the strong electricfield extending from the tip; the gas atoms lose an electron to the tip,and form positively charged noble gas ions.

The positively charged noble gas ions are accelerated away from the tip,and a certain fraction of the gas ions 1290 pass through extractor 1260and exit chamber 1210, into an ion optical column that includes lens1270, which further deflects and/or focuses the ions.

Electrode 1220 is tapered to increase the magnitude of the localelectric field in the region near the apex of the tip. Depending uponthe sharpness of the taper and the magnitude of potential V_(T), theregion of space in chamber 1210 within which ionization of noble gasatoms occurs can be relatively tightly controlled. As a result, arelatively well collimated beam of noble gas ions 1290 can be obtainedfollowing extractor 1260.

As discussed above in connection with hydrogen ions, the resulting beamof noble gas ions 1290 can be transported through a charged particleoptical column that includes various particle optical elements fordeflecting and/or focusing the noble gas ion beam. The noble gas ionbeam can also pass through an electrostatic and/or magnetic separator,as discussed above in connection with FIG. 44.

Noble gas ions that can be produced in field ionization generator 1200include helium ions, neon ions, argon ions, and krypton ions. Inaddition, field ionization generator 1200 can be used to generate ionsof other gaseous chemical species, including hydrogen, nitrogen, andoxygen.

Noble gas ions may have particular advantages relative to other ionspecies when treating biomass. For example, while noble gas ions canreact with biomass materials, neutralized noble gas ions (e.g., noblegas atoms) that are produced from such reactions are generally inert,and do not further react with the biomass. Moreover, neutral noble gasatoms do not remain embedded in the biomass material, but insteaddiffuse out of the material. Noble gases are non-toxic and can be usedin large quantities without adverse consequences to either human healthor the environment.

(iii) Carbon, Oxygen, and Nitrogen Ions

Ions of carbon, oxygen, and nitrogen can typically be produced by fieldionization in a system such as field ionization source 1100 or fieldionization generator 1200. For example, oxygen gas molecules and/oroxygen atoms (e.g., produced by heating oxygen gas) can be introducedinto a chamber, where the oxygen molecules and/or atoms are fieldionized to produce oxygen ions. Depending upon the sign of the potentialapplied to the field ionization electrode, positively- and/ornegatively-charged oxygen ions can be produced. The desired ion speciescan be preferentially selected from among various ion species andneutral atoms and molecules by an electrostatic and/or magnetic particleselector, as shown in FIG. 44.

As another example, nitrogen gas molecules can be introduced into thechamber of either field ionization source 1100 or field ionizationgenerator 1200, and ionized to form positively- and/ornegatively-charged nitrogen ions by the relatively strong electric fieldwithin the chamber. The desired ion species can then be separated fromother ionic and neutral species via an electrostatic and/or magneticseparator, as shown in FIG. 44.

To form carbon ions, carbon atoms can be supplied to the chamber ofeither field ionization source 1100 or field ionization generator 1200,wherein the carbon atoms can be ionized to form either positively-and/or negatively-charged carbon ions. The desired ion species can thenbe separated from other ionic and neutral species via an electrostaticand/or magnetic separator, as shown in FIG. 44. The carbon atoms thatare supplied to the chamber of either field ionization source 1100 orfield ionization generator 1200 can be produced by heating acarbon-based target (e.g., a graphite target) to cause thermal emissionof carbon atoms from the target. The target can be placed in relativelyclose proximity to the chamber, so that emitted carbon atoms enter thechamber directly following emission.

(iv) Heavier Ions

Ions of heavier atoms such as sodium and iron can be produced via anumber of methods. For example, in some embodiments, heavy ions such assodium and/or iron ions are produced via thermionic emission from atarget material that includes sodium and/or iron, respectively. Suitabletarget materials include materials such as sodium silicates and/or ironsilicates. The target materials typically include other inert materialssuch as beta-alumina. Some target materials are zeolite materials, andinclude channels formed therein to permit escape of ions from the targetmaterial.

FIG. 46 shows a thermionic emission source 1300 that includes a heatingelement 1310 that contacts a target material 1330, both of which arepositioned inside an evacuated chamber 1305. Heating element 1310 iscontrolled by controller 1320, which regulates the temperature ofheating element 1310 to control the ion current generated from targetmaterial 1330. When sufficient heat is supplied to target material 1330,thermionic emission from the target material generates a stream of ions1340. Ions 1340 can include positively-charged ions of materials such assodium, iron, and other relatively heavy atomic species (e.g., othermetal ions). Ions 1340 can then be collimated, focused, and/or otherwisedeflected by electrostatic and/or magnetic electrodes 1350, which canalso deliver ions 1340 to an injector.

Thermionic emission to form ions of relatively heavy atomic species isalso discussed, for example, in U.S. Pat. No. 4,928,033, entitled“Thermionic Ionization Source,” the entire contents of which areincorporated herein by reference.

In certain embodiments, relatively heavy ions such as sodium ions and/oriron ions can be produced by microwave discharge. FIG. 47 shows aschematic diagram of a microwave discharge source 1400 that producesions from relatively heavy atoms such as sodium and iron. Dischargesource 1400 includes a microwave field generator 1410, a waveguide tube1420, a field concentrator 1430, and an ionization chamber 1490. Duringoperation, field generator 1410 produces a microwave field whichpropagates through waveguide 1420 and concentrator 1430; concentrator1430 increases the field strength by spatially confining the field, asshown in FIG. 47. The microwave field enters ionization chamber 1490. Ina first region inside chamber 1490, a solenoid 1470 produces a strongmagnetic field 1480 in a region of space that also includes themicrowave field. Source 1440 delivers atoms 1450 to this region ofspace. The concentrated microwave field ionizes atoms 1450, and themagnetic field 1480 generated by solenoid 1470 confines the ionizedatoms to form a localized plasma. A portion of the plasma exits chamber1490 as ions 1460. Ions 1460 can then be deflected and/or focused by oneor more electrostatic and/or magnetic elements, and delivered to aninjector.

Atoms 1450 of materials such as sodium and/or iron can be generated bythermal emission from a target material, for example. Suitable targetmaterials include materials such as silicates and other stable salts,including zeolite-based materials. Suitable target materials can alsoinclude metals (e.g., iron), which can be coated on an inert basematerial such as a glass material.

Microwave discharge sources are also discussed, for example, in thefollowing U.S. Patents: U.S. Pat. No. 4,409,520, entitled “MicrowaveDischarge Ion Source,” and U.S. Pat. No. 6,396,211, entitled “MicrowaveDischarge Type Electrostatic Accelerator Having Upstream and DownstreamAcceleration Electrodes.” The entire contents of each of the foregoingpatents are incorporated herein by reference.

3. Particle Beam Sources

Particle beam sources that generate beams for use in irradiating biomassmaterial typically include three component groups: an injector, whichgenerates or receives ions and introduces the ions into an accelerator;an accelerator, which receives ions from the injector and increases thekinetic energy of the ions; and output coupling elements, whichmanipulate the beam of accelerated ions.

(i) Injectors

Injectors can include, for example, any of the ion sources discussed inthe preceding sections above, which supply a stream of ions forsubsequent acceleration. Injectors can also include various types ofelectrostatic and/or magnetic particle optical elements, includinglenses, deflectors, collimators, filters, and other such elements. Theseelements can be used to condition the ion beam prior to entering theaccelerator; that is, these elements can be used to control thepropagation characteristics of the ions that enter the accelerator.Injectors can also include pre-accelerating electrostatic and/ormagnetic elements that accelerate charged particles to a selected energythreshold prior to entering the accelerator. An example of an injectoris shown in Iwata, Y. et al., Alternating-Phase-Focused IH-DTL forHeavy-Ion Medical Accelerators”, Proceedings of EPAC 2006, Edinburgh,Scotland.

(ii) Accelerators

One type of accelerator that can be used to accelerate ions producedusing the sources discussed above is a Dynamitron® (available, forexample, from Radiation Dynamics Inc., now a unit of IBA,Louvain-la-Neuve, Belgium). A schematic diagram of a Dynamitron®accelerator 1500 is shown in FIG. 48. Accelerator 1500 includes aninjector 1510 (which includes an ion source), and an accelerating column1520 that includes a plurality of annular electrodes 1530. Injector 1510and column 1520 are housed within an enclosure 1540 that is evacuated bya vacuum pump 1600.

Injector 1510 produces a beam of ions 1580, and introduces beam 1580into accelerating column 1520. The annular electrodes 1530 aremaintained at different electric potentials, so that ions areaccelerated as they pass through gaps between the electrodes (e.g., theions are accelerated in the gaps, but not within the electrodes, wherethe electric potentials are uniform). As the ions travel from the top ofcolumn 1520 toward the bottom in FIG. 48, the average speed of the ionsincreases. The spacing between subsequent annular electrodes 1530typically increases, therefore, to accommodate the higher average ionspeed.

After the accelerated ions have traversed the length of column 1520, theaccelerated ion beam 1590 is coupled out of enclosure 1540 throughdelivery tube 1555. The length of delivery tube 1555 is selected topermit adequate shielding (e.g., concrete shielding) to be positionedadjacent to column 1520 to isolate the column. After passing throughtube 1555, ion beam 1590 passes through scan magnet 1550. Scan magnet1550, which is controlled by an external logic unit (not shown), cansweep accelerated ion beam 1590 in controlled fashion across atwo-dimensional plane oriented perpendicular to a central axis of column1520. As shown in FIG. 48, ion beam 1590 passes through window 1560(e.g., a metal foil window or screen) and then is directed to impinge onselected regions of a sample 1570 by scan magnet 1550.

In some embodiments, the electric potentials applied to electrodes 1530are static potentials generated, for example, by DC potential sources.In certain embodiments, some or all of the electric potentials appliedto electrodes 1530 are variable potentials generated by variablepotential sources. Suitable variable sources of large electricpotentials include amplified field sources such as klystrons, forexample. Accordingly, depending upon the nature of the potentialsapplied to electrodes 1530, accelerator 1500 can operate in eitherpulsed or continuous mode.

To achieve a selected accelerated ion energy at the output end of column1520, the length of column 1520 and the potentials applied to electrodes1530 are chosen based on considerations that are well-known in the art.However, it is notable that to reduce the length of column 1520,multiply-charged ions can be used in place of singly-charged ions. Thatis, the accelerating effect of a selected electric potential differencebetween two electrodes is greater for an ion bearing a charge ofmagnitude 2 or more than for an ion bearing a charge of magnitude 1.Thus, an arbitrary ion X²⁺ can be accelerated to a final energy E over ashorter length than a corresponding arbitrary ion X. Triply- andquadruply-charged ions (e.g., X³⁺ and X⁴⁺) can be accelerated to finalenergy E over even shorter distances. Therefore, the length of column1520 can be significantly reduced when ion beam 1580 includes primarilymultiply-charged ion species.

To accelerate positively-charged ions, the potential differences betweenelectrodes 1530 of column 1520 are selected so that the direction ofincreasing field strength in FIG. 48 is downward (e.g., toward thebottom of column 1520). Conversely, when accelerator 1500 is used toaccelerate negatively-charged ions, the electric potential differencesbetween electrodes 1530 are reversed in column 1520, and the directionof increasing field strength in FIG. 48 is upward (e.g., toward the topof column 1520). Reconfiguring the electric potentials applied toelectrodes 1530 is a straightforward procedure, so that accelerator 1500can be converted relatively rapidly from accelerating positive ions toaccelerating negative ions, or vice versa. Similarly, accelerator 1500can be converted rapidly from accelerating singly-charged ions toaccelerating multiply-charged ions, and vice versa.

Another type of accelerator that can be used to accelerate ions fortreatment of biomass-based material is a Rhodotron® accelerator(available, for example, from IBA, Louvain-la-Neuve, Belgium). Ingeneral, Rhodotron-type accelerators include a single recirculatingcavity through which ions that are being accelerated make multiplepasses. As a result, Rhodotron® accelerators can be operated incontinuous mode at relatively high continuous ion currents.

FIG. 49 shows a schematic diagram of a Rhodotron® accelerator 1700.Accelerator 1700 includes an injector 1710, which introduces acceleratedions into recirculating cavity 1720. An electric field source 1730 ispositioned within an inner chamber 1740 of cavity 1720, and generates anoscillating radial electric field. The oscillation frequency of theradial field is selected to match the transit time of injected ionsacross one pass of recirculating cavity 1720. For example, apositively-charged ion is injected into cavity 1720 by injector 1710when the radial electric field in the cavity has zero amplitude. As theion propagates toward chamber 1740, the amplitude of the radial field inchamber 1740 increases to a maximum value, and then decreases again. Theradial field points inward toward chamber 1740, and the ion isaccelerated by the radial field. The ion passes through a hole in thewall of inner chamber 1740, crosses the geometrical center of cavity1720, and passes out through another hole in the wall of inner chamber1740. When the ion is positioned at the enter of cavity 1720, theelectric field amplitude inside cavity 1720 has been reduced to zero (ornearly zero). As the ion emerges from inner chamber 1740, the electricfield amplitude in cavity 1720 begins to increase again, but the fieldis now oriented radially outward. The field magnitude during the secondhalf of the ion's pass through cavity 1720 again reaches a maximum andthen begins to diminish. As a result, the positive ion is againaccelerated by the electric field as the ion completes the second halfof a first pass through cavity 1720.

Upon reaching the wall of cavity 1720, the magnitude of the electricfield in cavity 1720 is zero (or nearly zero) and the ion passes throughan aperture in the wall and encounters one of beam deflection magnets1750. The beam deflection magnets essentially reverse the trajectory ofthe ion, as shown in FIG. 49, directing the ion to re-enter cavity 1720through another aperture in the wall of the chamber. When the ionre-enters cavity 1720, the electric field therein begins to increase inamplitude again, but is now once more oriented radially inward. Thesecond and subsequent passes of the ion through cavity 1720 follow asimilar pattern, so that the orientation of the electric field alwaysmatches the direction of motion of the ion, and the ion is acceleratedon every pass (and every half-pass) through cavity 1720.

As shown in FIG. 49, after six passes through cavity 1720, theaccelerated ion is coupled out of cavity 1720 as a portion ofaccelerated ion beam 1760. The accelerated ion beam passes through oneor more electrostatic and/or magnetic particle optical elements 1770,which can include lenses, collimators, beam deflectors, filters, andother optical elements. For example, under control of an external logicunit, elements 1770 can include an electrostatic and/or magneticdeflector that sweeps accelerated beam 1760 across a two-dimensionalplanar region oriented perpendicular to the direction of propagation ofbeam 1760.

Ions that are injected into cavity 1720 are accelerated on each passthrough cavity 1720. In general, therefore, to obtain accelerated beamshaving different average ion energies, accelerator 1700 can include morethan one output coupling. For example, in some embodiments, one or moreof deflection magnets 1750 can be modified to allow a portion of theions reaching the magnets to be coupled out of accelerator 1700, and aportion of the ions to be returned to chamber 1720. Multiple acceleratedoutput beams can therefore be obtained from accelerator 1700, each beamcorresponding to an average ion energy that is related to the number ofpasses through cavity 1720 for the ions in the beam.

Accelerator 1700 includes 5 deflection magnets 1750, and ions injectedinto cavity 1720 make 6 passes through the cavity. In general, however,accelerator 1700 can include any number of deflection magnets, and ionsinjected into cavity 1720 can make any corresponding number of passesthrough the cavity. For example, in some embodiments, accelerator 1700can include at least 6 deflection magnets and ions can make at least 7passes through the cavity (e.g., at least 7 deflection magnets and 8passes through the cavity, at least 8 deflection magnets and 9 passesthrough the cavity, at least 9 deflection magnets and 10 passes throughthe cavity, at least 10 deflection magnets and 11 passes through thecavity).

Typically, the electric field generated by field source 1730 provides asingle-cavity-pass gain of about 1 MeV to an injected ion. In general,however, higher single-pass gains are possible by providing ahigher-amplitude electric field within cavity 1720. In some embodiments,for example, the single-cavity-pass gain is about 1.2 MeV or more (e.g.,1.3 MeV or more, 1.4 MeV or more, 1.5 MeV or more, 1.6 MeV or more, 1.8MeV or more, 2.0 MeV or more, 2.5 MeV or more).

The single-cavity-pass gain also depends upon the magnitude of thecharge carried by the injected ion. For example, ions bearing multiplecharges will experience higher single-pass-cavity gain than ions bearingsingle charges, for the same electric field within cavity. As a result,the single-pass-cavity gain of accelerator 1700 can be further increasedby injecting ions having multiple charges.

In the foregoing description of accelerator 1700, a positively-chargedion was injected into cavity 1720. Accelerator 1700 can also acceleratenegatively charged ions. To do so, the negatively charged ions areinjected so that the direction of their trajectories is out of phasewith the radial electric field direction. That is, the negativelycharged ions are injected so that on each half pass through cavity 1720,the direction of the trajectory of each ion is opposite to the directionof the radial electric field. Achieving this involves simply adjustingthe time at which negatively-charged ions are injected into cavity 1720.Accordingly, accelerator 1700 is capable of simultaneously acceleratingions having the same approximate mass, but opposite charges. Moregenerally, accelerator 1700 is capable of simultaneously acceleratingdifferent types of both positively- and negatively-charged (and bothsingly- and multiply-charged) ions, provided that the transit times ofthe ions across cavity 1720 are relatively similar. In some embodiments,accelerator 1700 can include multiple output couplings, providingdifferent types of accelerated ion beams having similar or differentenergies.

Other types of accelerators can also be used to accelerate ions forirradiation of biomass material. For example, in some embodiments, ionscan be accelerated to relatively high average energies in cyclotron-and/or synchrotron-based accelerators. The construction and operation ofsuch accelerators is well-known in the art. As another example, in someembodiments, Penning-type ion sources can be used to generate and/oraccelerate ions for treating biomass-based material. The design ofPenning-type sources is discussed in section 7.2.1 of Prelec (KrstoPrelec, FIZIKA B 6 (1997) 4, 177-206).

Static and/or dynamic accelerators of various types can also generallybe used to accelerate ions. Static accelerators typically include aplurality of electrostatic lenses that are maintained at different DCvoltages. By selecting appropriate values of the voltages applied toeach of the lens elements, ions introduced into the accelerator can beaccelerated to a selected final energy. FIG. 50 shows a simplifiedschematic diagram of a static accelerator 1800 that is configured toaccelerate ions to treat biomass material 1835. Accelerator 1800includes an ion source 1810 that produces ions and introduces the ionsinto an ion column 1820. Ion column 1820 includes a plurality ofelectrostatic lenses 1825 that accelerate the ions generated by ionsource 1810 to produce an ion beam 1815. DC voltages are applied tolenses 1825; the potentials of the lenses remain approximately constantduring operation. Generally, the electrical potential within each lensis constant, and the ions of ion beam 1815 are accelerated in the gapsbetween the various lenses 1825. Ion column 1820 also includes adeflection lens 1830 and a collimation lens 1832. These two lensesoperate to direct ion beam 1815 to a selected position on biomassmaterial 1835, and to focus ion beam 1815 onto the biomass material.

Although FIG. 50 shows a particular embodiment of a static accelerator,many other variations are possible and suitable for treating biomassmaterial. In some embodiments, for example, the relative positions ofdeflection lens 1830 and collimation lens 1832 along ion column 1820 canbe exchanged. Additional electrostatic lenses can also be present in ioncolumn 1820, and ion column 1820 can further include magnetostaticoptical elements. In certain embodiments, a wide variety of additionalelements can be present in ion column 1820, including deflectors (e.g.,quadrupole, hexapole, and/or octopole deflectors), filtering elementssuch as apertures to remove undesired species (e.g., neutrals and/orcertain ionic species) from ion beam 1815, extractors (e.g., toestablish a spatial profile for ion beam 1815), and other electrostaticand/or magnetostatic elements.

Dynamic linear accelerators—often referred to as LINACs—can also be usedto generate an ion beam that can be used to treat biomass. Typically,dynamic linear accelerators include an ion column with a linear seriesof radiofrequency cavities, each of which produces an intense,oscillating radiofrequency (RF) field that is timed to coincide withinjection and propagation of ions into the ion column. As an example,devices such as klystrons can be used to generated the RF fields in thecavities. By matching the field oscillations to the injection times ofions, the RF cavities can accelerate ions to high energies withouthaving to maintain peak potentials for long periods of time. As aresult, LINACs typically do not have the same shielding requirements asDC accelerators, and are typically shorter in length. LINACs typicallyoperate at frequencies of 3 GHz (S-band, typically limited to relativelylow power) and 1 GHz (L-band, capable of significantly higher poweroperation). Typical LINACs have an overall length of 2-4 meters.

A schematic diagram of a dynamic linear accelerator 1850 (e.g., a LINAC)is shown in FIG. 51. LINAC 1850 includes an ion source 1810 and an ioncolumn 1855 that includes three acceleration cavities 1860, a deflector1865, and a focusing lens 1870. Deflector 1865 and focusing lens 1870function to steer and focus ion beam 1815 onto biomass material 1835following acceleration, as discussed above. Acceleration cavities 1860are formed of a conductive material such as copper, and function as awaveguide for the accelerated ions. Klystrons 1862, connected to each ofcavities 1860, generate the dynamic RF fields that accelerate the ionswithin the cavities. Klystrons 1862 are individually configured toproduce RF fields that, together, accelerate the ions in ion beam 1815to a final, selected energy prior to being incident on biomass material1835.

As discussed above in connection with static accelerators, manyvariations of dynamic accelerator 1850 are possible and can be used toproduce an ion beam for treating biomass material. For example, in someembodiments, additional electrostatic lenses can also be present in ioncolumn 1855, and ion column 1855 can further include magnetostaticoptical elements. In certain embodiments, a wide variety of additionalelements can be present in ion column 1855, including deflectors (e.g.,quadrupole, hexapole, and/or octopole deflectors), filtering elementssuch as apertures to remove undesired species (e.g., neutrals and/orcertain ionic species) from ion beam 1815, extractors (e.g., toestablish a spatial profile for ion beam 1815), and other electrostaticand/or magnetostatic elements. In addition to the specific static anddynamic accelerators discussed above, other suitable accelerator systemsinclude, for example: DC insulated core transformer (ICT) type systems,available from Nissin High Voltage, Japan; S-band LINACs, available fromL3-PSD (USA), Linac Systems (France), Mevex (Canada), and MitsubishiHeavy Industries (Japan); L-band LINACs, available from IotronIndustries (Canada); and ILU-based accelerators, available from BudkerLaboratories (Russia). In some embodiments, van de Graaff-basedaccelerators can be used to produce and/or accelerate ions forsubsequent treatment of biomass. FIG. 52 shows an embodiment of a van deGraaff accelerator 1900 that includes a spherical shell electrode 1902and an insulating belt 1906 that recirculates between electrode 1902 anda base 1904 of accelerator 1900. During operation, insulating belt 1906travels over pulleys 1910 and 1908 in the direction shown by arrow 1918,and carries charge into electrode 1902. Charge is removed from belt 1906and transferred to electrode 1902, so that the magnitude of theelectrical potential on electrode 1902 increases until electrode 1902 isdischarged by a spark (or, alternatively, until the charging current isbalanced by a load current).

Pulley 1910 is grounded, as shown in FIG. 52. A corona discharge ismaintained between a series of points or a fine wire on one side of belt1906. Wire 1914 is configured to maintain the corona discharge inaccelerator 1900. Wire 1914 is maintained at a positive potential, sothat belt 1906 intercepts positive ions moving from wire 1914 to pulley1910. As belt 1906 moves in the direction of arrow 1918, the interceptedcharges are carried into electrode 1902, where they are removed frombelt 1906 by a needle point 1916 and transferred to electrode 1902. As aresult, positive charges accumulate on the surface of electrode 1902;these charges can be discharged from the surface of electrode 1902 andused to treat biomass material. In some embodiments, accelerator 1900can be configured to provide negatively charged ions by operating wire1914 and needle point 1916 at a negative potential with respect togrounded pulley 1910.

In general, accelerator 1900 can be configured to provide a wide varietyof different types of positive and negative charges for treatingbiomass. Exemplary types of charges include electrons, protons, hydrogenions, carbon ions, oxygen ions, halogen ions, metal ions, and othertypes of ions.

In certain embodiments, tandem accelerators (including folded tandemaccelerators) can be used to generate ion beams for treatment of biomassmaterial. An example of a folded tandem accelerator 1950 is shown inFIG. 53. Accelerator 1950 includes an accelerating column 1954, a chargestripper 1956, a beam deflector 1958, and an ion source 1952.

During operation, ion source 1952 produces a beam 1960 of negativelycharged ions, which is directed to enter accelerator 1950 through inputport 1964. In general, ion source 1952 can be any type of ion sourcethat produces negatively charged ions. For example, suitable ion sourcesinclude a source of negative ions by cesium sputtering (SNICS) source, aRF-charge exchange ion source, or a toroidal volume ion source (TORVIS).Each of the foregoing exemplary ion sources is available, for example,from National Electrostatics Corporation (Middleton, Wis.).

Once inside accelerator 1950, the negative ions in beam 1960 areaccelerated by accelerating column 1954. Typically, accelerating column1954 includes a plurality of accelerating elements such as electrostaticlenses. The potential difference applied in column 1954 to acceleratethe negative ions can be generated using various types of devices. Forexample, in some embodiments, (e.g., PELLERTRON® accelerators), thepotential is generated using a PELLERTRON® charging device. PELLERTRON®devices include a charge-carrying belt that is formed from a pluralityof metal (e.g., steel) chain links or pellets that are bridged byinsulating connectors (e.g., formed from a material such as nylon).During operation, the belt recirculates between a pair of pulleys, oneof which is maintained at ground potential. As the belt moves betweenthe grounded pulley and the opposite pulley (e.g., the terminal pulley),the metal pellets are positively charged by induction. Upon reaching theterminal pulley, the positive charge that has accumulated on the belt isremoved, and the pellets are negatively charged as they leave theterminal pulley and return to the ground pulley.

The PELLERTRON® device generates a large positive potential withincolumn 1954 that is used to accelerate the negative ions of beam 1960.After undergoing acceleration in column 1954, beam 1960 passes throughcharge stripper 1956. Charge stripper 1956 can be implemented as a thinmetal foil and/or a tube containing a gas that strips electrons from thenegative ions, for example. The negatively charged ions are therebyconverted to positively charged ions, which emerge from charge stripper1956. The trajectories of the emerging positively charged ions arealtered so that the positively charged ions travel back throughaccelerating column 1954, undergoing a second acceleration in the columnbefore emerging as positively charged ion beam 1962 from output port1966. Positively charged ion beam 1962 can then be used to treat biomassmaterial according to the various methods disclosed herein.

Due to the folded geometry of accelerator 1950, ions are accelerated toa kinetic energy that corresponds to twice the potential differencegenerated by the PELLERTRON® charging device. For example, in a 2 MVPELLERTRON® accelerator, hydride ions that are introduced by ion source1952 will be accelerated to an intermediate energy of 2 MeV during thefirst pass through column 1954, converted to positive ions (e.g.,protons), and accelerated to a final energy of 4 MeV during the secondpass through column 1954.

In certain embodiments, column 1954 can include elements in addition to,or as alternatives to, the PELLERTRON® charging device. For example,column 1954 can include static accelerating elements (e.g., DCelectrodes) and/or dynamic acceleration cavities (e.g., LINAC-typecavities with pulse RF field generators for particle acceleration).Potentials applied to the various accelerating devices are selected toaccelerate the negatively charged ions of beam 1960.

Exemplary tandem accelerators, including both folded and non-foldedaccelerators, are available from National Electrostatics Corporation(Middleton, Wis.), for example.

In some embodiments, combinations of two or more of the various types ofaccelerators can be used to produce ion beams that are suitable fortreating biomass. For example, a folded tandem accelerator can be usedin combination with a linear accelerator, a Rhodotron® accelerator, aDynamitron®, a static accelerator, or any other type of accelerator toproduce ion beams. Accelerators can be used in series, with the outpution beam from one type of accelerator directed to enter another type ofaccelerator for additional acceleration. Alternatively, multipleaccelerators can be used in parallel to generate multiple ion beams forbiomass treatment. In certain embodiments, multiple accelerators of thesame type can be used in parallel and/or in series to generateaccelerated ion beams.

In some embodiments, multiple similar and/or different accelerators canbe used to generate ion beams having different compositions. Forexample, a first accelerator can be used to generate one type of ionbeam, while a second accelerator can be used to generate a second typeof ion beam. The two ion beams can then each be further accelerated inanother accelerator, or can be used to treat biomass.

Further, in certain embodiments, a single accelerator can be used togenerate multiple ion beams for treating biomass. For example, any ofthe accelerators discussed herein (and other types of accelerators aswell) can be modified to produce multiple output ion beams bysub-dividing an initial ion current introduced into the accelerator froman ion source. Alternatively, or in addition, any one ion beam producedby any of the accelerators disclosed herein can include only a singletype of ion, or multiple different types of ions.

In general, where multiple different accelerators are used to produceone or more ion beams for treatment of biomass, the multiple differentaccelerators can be positioned in any order with respect to one another.This provides for great flexibility in producing one or more ion beams,each of which has carefully selected properties for treating biomass(e.g., for treating different components in biomass).

The ion accelerators disclosed herein can also be used in combinationwith any of the other biomass treatment steps disclosed herein. Forexample, in some embodiments, electrons and ions can be used incombination to treat biomass. The electrons and ions can be producedand/or accelerated separately, and used to treat biomass sequentially(in any order) and/or simultaneously. In certain embodiments, electronand ion beams can be produced in a common accelerator and used to treatbiomass. For example, many of the ion accelerators disclosed herein canbe configured to produce electron beams as an alternative to, or inaddition to, ion beams. For example, Dynamitron® accelerators,Rhodotron® accelerators, and LINACs can be configured to produceelectron beams for treatment of biomass.

Moreover, treatment of biomass with ion beams can be combined with othertechniques such as sonication. In general, sonication-based treatmentcan occur before, during, or after ion-based biomass treatment. Othertreatments such as electron beam treatment can also occur in anycombination and/or order with ultrasonic treatment and ion beamtreatment.

(iii) Output Coupling Elements and Other Components

In general, any of the sources disclosed herein can include varioustypes of output coupling elements to control the propagation andcharacteristics of accelerated ion beams. For example, sources caninclude one or more ion lenses, deflectors, filters, collimators, orother electrode-based elements, to which both static and variablepotentials can be applied. These elements can be electrostatic,magnetic, or both electrostatic and magnetic.

Sources can include one or more electric and/or magnetic field sources,including static field sources and/or variable field sources. Variablefield sources can produce fields having frequencies ranging from 1 Hz to10¹⁵ Hz.

In some embodiments, ozone is produced when accelerated ions interactwith atmospheric oxygen gas. Production of excess ozone gas mayrepresent a potential health hazard to system operators working in thevicinity of the sources disclosed herein. Accordingly, the sources caninclude an ozone removal system, which typically includes one or moreoutlet vents connected to vacuum pumps to actively remove ozone andother gases. In certain embodiments, sources can include a shield thanencloses a volume of space through which the accelerated ions travel, toassist in confining ozone gas to the enclosed volume. The enclosedvolume can be pumped by an evacuation system.

In some embodiments, accelerated ions are used to directly treat biomassmaterial. However, due to the relatively sharp Bragg peak in the dosedistribution for many types of ions, providing uniform treatment ofthick materials can be challenging. Accordingly, in some embodiments,when relatively thick biomass material is treated with an acceleratedion beam, the energy of the ion beam is changed during exposure of thematerial (for example, by changing certain accelerating potentials in anaccelerator). The effect of changing the energy of the ion beam is to“sweep” the Bragg peak of the dose distribution through the thickness ofthe material. The sweeping of the Bragg peak can be performed in amanner such that the ion dose received throughout the thickness of thematerial is nominally uniform.

In certain embodiments, a similar effect can be achieved by spreadingout the Bragg peak of the ion beam. For example, a dispersive elementcan be placed in the path of the accelerated ions to cause broadening ofthe energy spectrum of the accelerated ions, as shown in FIG. 2 of Chu(2006). As a result of the energy broadening, the Bragg peak can besignificantly broadened, resulting in more uniform dosing of an exposedbiomass material.

In certain embodiments, charged particles used to expose biomassmaterials can include antiparticles. For example, in some embodiments,antiparticles such as positrons and/or antiprotons can be used to exposematerials. Moreover, in certain embodiments, various different isotopesof ions can be used to expose biomass materials. For example, deuteriumions and/or ions derived from various isotopes of carbon, nitrogen,oxygen, and various metals, can be used. In particular, in someembodiments, an ion beam that exposes biomass materials can include atleast some ions that are positron emitters, such as ions of ¹⁰C, ¹¹C,and ¹⁵O. When these ions interact with material such as biomassmaterial, the ions emit positrons in a region of the material close tothe position of the Bragg peak. By monitoring positron emission, theposition of the Bragg peak can therefore be located in the material.This technique can be particularly useful when the Bragg peak is sweptthrough the material by changing the ion energy, as discussed above.

In some embodiments, combinations of different ions can be used to treatbiomass material. For example, material can be treated with acombination of protons and carbon ions. In general, any combination oftwo or more ions can be used to treat material; the ions have the sameor different charge signs and magnitudes, and the same or differentmasses. Different ions can, in certain embodiments, be accelerated inthe same accelerator. Alternatively, or in addition, different ions canbe accelerated in different accelerators, and biomass treatment facilitycan include multiple ion accelerators configured to produce ion beams.

4. Operating Parameters

In general, when a condensed medium is exposed to a charged particlebeam, the charged particles penetrate the medium and deposit within themedium at a distribution of depths below the surface upon which theparticles are incident. It has generally been observed (see, forexample, FIG. 1 in Prelec (infra, 1997)) that the dose distribution forions includes a significantly sharper maximum (the Bragg peak), and thations exhibit significantly less lateral scattering, than other particlessuch as electrons and neutrons and other forms of electromagneticradiation such as x-rays. Accordingly, due to the relativelywell-controlled dosing profile of accelerated ions, they operaterelatively efficiently to alter the structure of biomass material.Furthermore, as is apparent from FIG. 6 of Prelec (infra, 1997), heavierions (such as carbon ions) have even sharper dosing profiles thanlighter ions such as protons, and so the relative effectiveness of theseheavier ions at treating biomass material is even greater than forlighter ions.

In some embodiments, the average energy of the accelerated ions that areincident on biomass material is 1 MeV/u or more (e.g., 2, 3, 4, 5, 6, 7,8, 9, 10, 12, 15, 20, 30, 50, 100, 300, 500, 600, 800, or even 1000MeV/u or more).

In certain embodiments, the average energy of the accelerated ions is 10MeV or more (e.g., 20, 30, 50, 100, 200, 300, 400, 500, 600, 800, 1000,2000, 3000, 4000, or even 5000 MeV or more).

In certain embodiments, an average velocity of the accelerated ions is0.0005 c or more (e.g., 0.005 c or more, 0.05 c or more, 0.1 c or more,0.2 c or more, 0.3 c or more, 0.4 c or more, 0.5 c or more, 0.6 c ormore, 0.7 c or more, 0.8 c or more, 0.9 c or more). In general, for agiven accelerating potential, lighter ions are accelerated to highervelocities than heavier ions. For example, for a given acceleratingpotential, a maximum velocity of a hydrogen ion may be about 0.05 c,while a maximum velocity of a carbon ion may be about 0.0005 c. Thesevalues are only exemplary; the velocity of the accelerated ions dependson the accelerating potential applied, the mode of operation of theaccelerator, the number of passes through the accelerating field, andother such parameters.

In some embodiments, an average ion current of the accelerated ions is10⁵ particles/s or more (e.g., 10⁶, 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, 10¹²,10¹³, 10¹⁴, 10¹⁵, or even 10¹⁶ particles/s or more).

In some embodiments, a radiation dose delivered to biomass material froman ion beam is 5 Mrad or more (e.g., 10, 15, 20, 30, 40, 50, 60, 80, oreven 100 Mrad or more).

5. Ion Beam Exposure Conditions

When a sample is exposed to an ion beam, energy is deposited in thesample according to an ion dose profile (also sometimes referred to as adepth-dose distribution). FIG. 54 shows a schematic diagram of arepresentative ion dose profile 2010 for a condensed-phase sample. Thevertical axis of ion dose profile 2010 in FIG. 54 shows the relative iondose, plotted as a function of depth below a surface of the sample thatis exposed to the ion beam, on the horizontal axis. FIG. 54 alsoincludes, for comparative purposes, an electron dose profile 2020, agamma radiation dose profile 2030, and an x-ray dose profile 2040.

As shown in FIG. 54, both gamma radiation and x-ray radiation (andfurther, other types of electromagnetic radiation) are absorbed stronglyin a region adjacent to the surface of the sample, leading to thehighest energy doses being deposited near the sample surface. Gamma andx-ray radiation dose profiles 2030 and 2040 decrease approximatelyexponentially from the surface of the sample, as progressively fewerphotons are able to penetrate deeper into the sample to be absorbed.

Electron dose profile 2020 shows a build-up effect whereby, due to thepenetrating ability of Compton electrons, the deposited energy doseincreases in the vicinity of the exposed surface of the sample to amaximum deposited dose at a penetration depth of, typically, about 3-4cm in condensed media. Thereafter, the relative dose of deposited energydecreases relatively rapidly with increasing distance beneath the samplesurface.

Ion beams, in contrast, typically have dose profiles that are sometimesdescribed as being inverse with respect to the dose profiles ofelectrons and photons. As shown in FIG. 54, ion dose profile 2010includes a region 2012 in which a relatively constant energy dose isapplied to the sample. Thereafter, ion dose profile 2010 includes aregion 2014 referred to as the Bragg peak, which corresponds to aportion of the sample into which a comparatively larger fraction of theion beam's energy is deposited, followed by a region 2016 in which amuch smaller energy dose is deposited. The Bragg peak, which has a fullwidth at half maximum (FWHM) of 6, ensures that the dose profile forions differs significantly from the dose profiles for electrons andphotons of various wavelengths. As a result, exposing materials such asbiomass materials to ion beams can yield effects that are different fromthe effects produced by photons and electron beams.

Typically, the width δ of Bragg peak 2014 depends upon a number offactors, including the nature of the sample, the type of ions, and theaverage ion energy. One important factor that influences the width δ ofBragg peak 2014 is the distribution of energies in the incident ionbeam. In general, the narrower the distribution of energies in theincident ion beam, the narrower the width δ of Bragg peak 2014. As anexample, Bragg peak 2014 typically has a width of about 3 mm or less fora distribution of ion energies that has a FWHM of 1 keV or less. Thewidth δ of Bragg peak 2014 can be much less than 3 mm under theseconditions as well, e.g., 2.5 mm or less, 2.0 mm or less, 1.5 mm orless, 1.0 mm or less.

The position of Bragg peak 2014, indicated by γ in FIG. 54, depends upona number of factors including the average energy of the incident ionbeam. In general, for larger average ion beam energies, Bragg peak 2014will shift to larger depths in FIG. 54, because higher-energy ions havethe ability to penetrate more deeply into a material before most of theions' kinetic energy is lost via scattering events.

Various properties of one or more incident ion beams can be adjusted toexpose samples (e.g., biomass materials) to ion beam radiation, whichcan lead to de-polymerization and other chain-scission reactions in thesamples, reducing the molecular weight of the samples in a predictableand controlled manner. FIG. 55 shows a schematic diagram of an ion beamexposure system 2100. System 2100 includes an ion source 2110 thatgenerates an ion beam 2150. Optical elements 2120 (including, forexample, lenses, apertures, deflectors, and/or other electrostaticand/or magnetic elements for adjusting ion beam 2150) direct ion beam2150 to be incident on sample 2130, which has a thickness h in adirection normal to surface 2135 of sample 2130. In addition todirecting ion beam 2150, optical elements 2120 can be used to controlvarious properties of ion beam 2150, including collimation and focusingof ion beam 2150. Sample 2130 typically includes, for example, one ormore of the various types of biomass materials that are discussedherein. System 2100 also includes an electronic controller 2190 inelectrical communication with the various components of the system (andwith other components not shown in FIG. 55). Electronic controller 2190can control and/or adjust any of the system parameters disclosed herein,either fully automatically or in response to input from a humanoperator.

FIG. 55 also shows the ion dose profile that results from exposure ofsample 2130 to ion beam 2150. The position 2160 of the Bragg peak withinsample 2130 depends upon the average energy of ion beam 2150, the natureof the ions in ion beam 2150, the material from which sample 2130 isformed, and other factors.

In many applications of ion beams, such as ion therapy for tumoreradication, the relatively small width δ of Bragg peak 2014 isadvantageous, because it allows reasonably fine targeting of particulartissues within a patient undergoing therapy, and helps to reduce damagedue to exposure of nearby benign tissues.

However, when exposing biomass materials such as sample 2130 to ion beam2150, the relatively small width δ of Bragg peak 2014 can restrictthroughput. Typically, for example, the thickness h of sample 2130 islarger than the width δ of Bragg peak 2014. In some embodiments, h canbe substantially larger than 6 (e.g., larger by a factor of 5 or more,or 10 or more, or 20 or more, or 50 or more, or 100 or more, or evenmore).

To increase a thickness of sample 2130 in which a selected dose can bedelivered in a particular time interval, the energy distribution of ionbeam 2150 can be adjusted. Various methods can be used to adjust theenergy distribution of ion beam 2150. One such method is to employ oneor more removable scattering elements 2170 positioned in the patch ofion beam 2150, as shown in FIG. 55. Scattering element 2170 can be, forexample, a thin membrane formed of a metal material such as tungsten,tantalum, copper, and/or a polymer-based material such as Lucite®.

Prior to passing through scattering element 2170, ion beam 2150 has anenergy distribution of width w, shown in FIG. 56A. When ion beam 2150passes through element(s) 2170, at least some of the ions in ion beam2150 undergo scattering events with atoms in element(s) 2170,transferring a portion of their kinetic energy to the atoms ofelement(s) 2170. As a result, the energy distribution of ion beam 2150is broadened to a width b larger than w, as shown in FIG. 56B. Inparticular, the energy distribution of ion beam 2150 acquires a broaderlow-energy tail as a result of scattering in element(s) 2170.

FIG. 56C shows the effect of broadening the ion energy distribution ofion beam 2150 on the ion dose profiles in sample 2130. Ion dose profile2140 a is produced by exposing sample 2130 to ion beam 2150 having theion energy distribution shown in FIG. 56A. Ion dose profile 2140 aincludes a relatively narrow Bragg peak. As a result, the region ofsample 2130 in which a relatively high dose is deposited is small. Incontrast, by broadening the ion energy distribution of ion beam 2150 toyield the distribution shown in FIG. 56B, ion dose profile 2140 b isobtained in sample 2130 after exposing the sample to the broadeneddistribution of ion energies. As dose profile 2140 b shows, bybroadening the ion energy distribution, the region of sample 2130 inwhich a relatively high dose is deposited is increased relative to iondose profile 2140 a. By increasing the region of sample 2130 exposed toa relatively high dose, the throughput of the exposure process can beimproved.

In certain embodiments, the width b of the broadened energy distributioncan be larger than w by a factor of 1.1 or more (e.g., 1.2, 1.3, 1.4,1.5, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, or even 10.0 or more).

Typically, the ion dose profile in sample 2130 produced by exposure ofthe sample to the broadened ion energy distribution shown in FIG. 56Bhas a Bragg peak having a full width at half maximum (FWHM) of ε. As aresult of broadening the ion energy distribution, ε can be larger than δby a factor of 1.1 or more (e.g., 1.2 or more, 1.3 or more, 1.5 or more,1.7 or more, 2.0 or more, 2.5 or more, 3.0 or more, 4.0 or more, 5.0 ormore, 6.0 or more, 7.0 or more, 10.0 or more).

For sample 2130 of thickness h, after broadening the ion energydistribution of ion beam 2150 and exposing the sample to the ion beam, aratio of ε/h can be 1×10⁻⁶ or more (e.g., 1×10⁻⁵, 5×10⁻⁵, 1×10⁻⁴,5×10⁻⁴, 1×10⁻³, 5×10⁻³, 0.01, 0.05, 0.08, 0.1, or even 0.5 or more).

In certain embodiments, sample 2130 includes a plurality of particles(e.g., approximately spherical particles, and/or fibers, and/orfilaments, and/or other particle types). In general, the particles havea distribution of different sizes, with an average particle size r. Theion energy distribution of ion beam 2150 can be adjusted (e.g., viabroadening) based on the average particle size r of sample 2130 toimprove the efficiency of ion-based treatment of sample 2130. Forexample, ion beam 2150 can be adjusted to that a ratio of 8/r is 0.001or more (e.g., 0.005 or more, 0.01 or more, 0.05 or more, 0.1 or more,0.5 or more, 1.0 or more, 1.5 or more, 2.0 or more, 2.5 or more, 3.0 ormore, 3.5 or more, 4.0 or more, 5.0 or more, 6.0 or more, 8.0 or more,10 or more, 50 or more, 100 or more, 500 or more, 1000 or more, or evenmore).

In some embodiments, a scattering element 2170 can include multipledifferent scattering sub-elements that are configured to broaden thedistribution of ion energies in ion beam 2150 by different amounts. Forexample, FIG. 57 shows a multi-sub-element scattering element 2170 thatincludes sub-elements 2170 a-e. Each of sub-elements 2170 a-e broadensthe distribution of ion energies in ion beam 2150 to a different extent.During operation of system 2100, electronic controller 2190 can beconfigured to select an appropriate sub-element of scattering element2170 based on information such as the thickness h of sample 2130, thetype of ions in ion beam 2150, and the average ion energy in ion beam2150. The selection of an appropriate sub-element can be made in fullyautomated fashion, or based at least in part on input from a humanoperator. Selection of an appropriate sub-element is made by translatingscattering element 2170 in the direction shown by arrow 2175 to positiona selected sub-element in the path of ion beam 2150.

In certain embodiments, other devices can be used in addition to, or asan alternative to, scattering element(s) 2170. For example, in someembodiments, combinations of electric and or magnetic fields, producedby ion optical elements, can be used to broaden the ion energydistribution of ion beam 2150. Ion beam 2150 can pass through a firstfield configured to spatially disperse ions in the ion beam. Then thespatially dispersed ions can pass through a second field that iswell-localized spatially, and which selectively retards only a portionof the spatially dispersed ions. The ions then pass through a thirdfield that spatially re-assembles all of the ions into a collimatedbeam, which is then directed onto the surface of sample 2130. Typically,the ion optical elements used to generate the fields that adjust the ionenergy distribution are controlled by electronic controller 2190. Byapplying spatially localized fields selectively, a high degree ofcontrol over the modified ion energy distribution is possible, includingthe generation of ion energy distributions having complicated profiles(e.g., multiple lobes). For example, in some embodiments, by applying alocalized field that accelerates a portion of the spatially dispersedion distribution, the ion energy distribution shown in FIG. 56A can bebroadened on the high-energy side of the distribution maximum.

The information used by electronic controller 2190 to adjust the ionenergy distribution of ion beam 2150 can include the thickness h ofsample 2130, as discussed above. In some embodiments, electroniccontroller 2190 can use information about the expected ion dose profilein sample 2130 to adjust the ion energy distribution of ion beam 2150.Information about the expected ion dose profile can be obtained from adatabase, for example, that includes measurements of ion dose profilesacquired from literature sources and/or from calibration experimentsperformed on representative samples of the material from which sample2130 is formed. Alternatively, or in addition, information about theexpected ion dose profile can be determined from a mathematical model ofion interactions in sample 2130 (e.g., an ion scattering model).

In certain embodiments, the information about the expected ion doseprofile can include information about the FWHM of the Bragg peak in theexpected ion dose profile.

The FWHM of the Bragg peak can be determined from measurements of iondose profiles and/or from one or more mathematical models of ionscattering in the sample. Adjustments of the ion energy distribution ofion beam 2150 can be performed to reduce a difference between thethickness h of sample 2130 and the FWHM of the Bragg peak. In someembodiments, for example, a difference between h and the full width athalf maximum of the Bragg peak is 20 cm or less (e.g., 18, 16, 14, 12,10, 8, 6 cm, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or even 0.001 cm orless).

In some embodiments, the ion beam exposure system can adjust thedistribution of ion energies in ion beam 2150 in other ways. Forexample, the ion beam exposure system can be configured to filter theion beam by removing ions from ion beam 2150 that have energies below aselected energy threshold and/or above a selected energy threshold. FIG.58 shows an ion beam exposure system 2200 that includes an ion filter2210 discussed in more detail below. The other components of system 2200are similar to the components of system 2100, and will not be furtherdiscussed.

FIG. 59A shows an ion energy distribution corresponding to ion beam 2150produced by ion source 2110. Ion beam 2150, with an energy distributionas shown in FIG. 59A, enters ion filter 2210 where the energydistribution of ion beam 2150 is adjusted by filtering out certain ionsfrom the ion beam. For example, in some embodiments, ion filter 2210 canbe configured to remove ions from ion beam 2150 that have an energysmaller than a selected energy threshold. In FIG. 59A, the selectedenergy threshold is the position E₀ of the peak in the ion energydistribution, although more generally, any energy threshold can beselected. By filtering out all (or even just a large fraction of) ionshaving an energy less than E₀, the ion energy distribution for ion beam2150 is as shown in FIG. 59B.

In contrast, in some embodiments, ion filter 2210 can be configured toremove ions from ion beam 2150 that have an energy larger than aselected energy threshold (e.g., when ion filter 2210 is implemented asa hemispherical analyzer). For example, the selected energy thresholdcan correspond to the position E₀ of the peak in the ion energydistribution, although more generally, any energy threshold can beselected. By removing all (or even a large fraction of) ions from ionbeam 2150 having an energy more than E₀, the ion energy distribution forion beam 2150 is as shown in FIG. 59C.

In certain embodiments, sample 2130 can be exposed directly to afiltered ion beam 2150. By filtering the ion beam to achieve a narrowerion energy distribution, for example, the ion dose profile in sample2130 is sharper following sample exposure than it would otherwise havebeen without filtering ion beam 2150. As a result, the width of theBragg peak in sample 2130 is smaller relative to the Bragg peak widthfor an unfiltered ion beam. By exposing sample 2130 to a narrowerdistribution of incident ion energies, more refined control over theposition of ion beam 2150 can be achieved; this level of ion exposurecontrol can be useful when exposing various types of delicate samplematerials.

Alternatively, the filtered ion beam can then be passed through one ormore scattering elements and/or other devices to increase the width ofthe distribution of ion energies. This two-step approach to modifyingthe ion energy distribution—a first filtering step, followed by a secondbroadening step—can be used to produce ion energy distributions that aretailored for specific applications (e.g., specific to certain ion types,certain materials, and/or certain pre-processing conditions) that maynot be achievable using a simpler one-step energy distributionbroadening procedure.

As an example, by first filtering ion beam 2150, and then passing thefiltered ion beam through one or more scattering elements 2170, theshape of the ion energy distribution can be made more Gaussian thanwould otherwise be possible using only a scattering step instead of thetwo-step procedure.

Ion filter 2210 can include one or more of a variety of differentdevices for removing ions from ion beam 2150. For example, in someembodiments, ion filter 2210 includes a hemispherical analyzer andaperture filter. The hemispherical analyzer includes a magnetic fieldsource that disperses the ions of ion beam 2150 according to theirkinetic energies. The aperture filter is then positioned in the path ofthe dispersed ion beam 2150 to permit only ions having a particularrange of energies to pass through the aperture.

In certain embodiments, other devices can be used to filter ion beam2150. For example, absorbing elements (e.g., elements configured toabsorb incident ions having energies smaller than a selected energythreshold can be used to filter ion beam 2150. Suitable absorbingelements include metal foils, for example.

In some embodiments, ion beam 2150 (and in particular, the Bragg peak inan expected ion dose profile produced following exposure of sample 2130to ion beam 2150) can be swept through sample 2130 to deliver selectedradiation doses to various portions of the sample. In general, theposition of the Bragg peak in sample 2130 can be selected by adjustingthe average energy of ion beam 2150 (the average energy of ion beam 2150typically corresponds to the maximum in the ion energy distribution).Ion source 2110, under the control of electronic controller 2190, canadjust the average energy of ion beam 2150 by changing an extractionvoltage applied to accelerate ions in the ion source.

FIG. 60 is a schematic diagram that shows how the Bragg peak of an iondose profile in sample 2130 can be swept through the sample. As a firststep, ion exposure system 2100 is configured to produce a first ion beamwith a selected average ion energy corresponding to a particularextraction voltage applied in ion source 2110. When sample 2130 isexposed to the first ion beam, ion dose profile 2010 a results in thesample, with the Bragg peak at position 2230 a. Following exposure, theextraction voltage in ion source 2110 is adjusted to produce a secondion beam with a different average ion energy. When sample 2130 isexposed to the second ion beam, ion dose profile 2010 b results in thesample. By further repeating the adjusting of the extraction voltage inion source 2110 to produce additional beams with different average ionenergies, and exposing sample 2130 to the additional beams, the Braggpeak of the ion dose profile can be swept through sample 2130 in thedirection shown by arrow 2220, for example. More generally, however, bychanging the extraction voltage in ion source 2110, the position of theBragg peak in sample 2130 can be selected as desired, permittingdelivery of large doses to selected regions of sample 2130 in anysequence.

In general, other properties of ion beam 2150 can also be adjusted inaddition to, or as an alternative to, adjusting the average ion energyof the ion beam. For example, in some embodiments, the divergence angleof ion beam 2150 at the surface of sample 2130 can be adjusted tocontrol the ion dose profile in sample 2130. Generally, by increasingthe divergence angle of ion beam 2150 at the surface of sample 2130, thefull width at half maximum of the Bragg peak in sample 2130 can beincreased. Thus, in certain embodiments, the average energy of the ionbeam can be maintained, but the ion dose profile in thematerial—including the position of the Bragg peak—can be changed byadjusting the ion beam's divergence angle.

The divergence angle can be adjusted automatically or by operatorcontrol by electronic controller 2190. Typically optical elements 2120include one or more ion beam steering elements such as quadrupole and/oroctopole deflectors. By adjusting potentials applied to the variouselectrodes of such deflectors, the divergence angle (and the angle ofincidence) of ion beam 2150 at the surface of sample 2130 can beadjusted.

In some embodiments—unlike in other applications of ion beams such assurgical intervention—it can be advantageous to use ion beams withrelatively large divergence angles, to ensure that the Bragg peakpositioned in sample 2130 covers a suitable fraction of the thickness ofsample 2130. For example, in certain embodiments, sample 2130 can beexposed to an ion beam having a divergence angle of 2 degrees or more(e.g., 5, 10, 15, 20, 30, 40, or even 50 degrees or more).

In some embodiments, both an ion beam current of ion beam 2150 and theaverage ion energy of ion beam 2150 can be adjusted to deliver arelatively constant dose as a function of thickness h of sample 2130.For example, if sample 2130 is exposed according to the sequential iondose profiles 2010 a, 2010 b, and 2010 c in FIG. 60, the net ion doseprofile in sample 2130 corresponds to the sum of profiles 2010 a-c,which is shown in FIG. 61A. Based on the net ion dose profile of FIG.61A, it is evident that certain regions of sample 2130 receive largernet doses than other regions of sample 2130.

The differences in net dose can be reduced by adjusting the ion beamcurrent of ion beam 2150 together with adjustments of the average ionenergy. The ion beam current can be adjusted in ion source 2110 underthe control of electronic controller 2190. For example, to reduce thedifference in the net dose delivered to sample 2130 when the Bragg peakis swept through sample 2130 in the direction indicated by arrow 2220 inFIG. 60, the ion beam current can be successively reduced for eachsuccessive reduction in ion beam energy. Three ion dose profiles, eachcorresponding to successive decreases in both average ion energy and ioncurrent in ion beam 2150, are shown as profiles 2010 d-f, respectively,in FIG. 61B. The net ion dose profile in sample 2130 that results fromthese three sequential exposures is shown in FIG. 61C. The net ion doseprofile shows significantly reduced variation as a function of positionin sample 2130 relative to the net ion dose profile of FIG. 61A.

By carefully controlling the average energy and ion current of ion beam2150, variations in net relative ion dose through the thickness ofsample 2130 following exposure of the sample to ion beam 2150 can berelatively small. For example, a difference between a maximum netrelative ion dose and a minimum net relative ion dose in sample 2130following multiple exposures to ion beam 2150 can be 0.2 or less (e.g.,0.15, 0.1, 0.05, 0.04, 0.03, 0.02, 0.01 or even 0.005 or less).

By controlling the average energy and ion current of ion beam 2150, eachportion of the exposed sample can receive a net dose of between 0.001Mrad and 100 Mrad following multiple exposures to the ion beam (e.g.,between 0.005 Mrad and 50 Mrad, between 0.01 Mrad and 50 Mrad, between0.05 Mrad and 30 Mrad, between 0.1 Mrad and 20 Mrad, between 0.5 Mradand 20 Mrad, between 1 Mrad and 10 Mrad).

In some embodiments, sample 2130 can be exposed to different types ofions. Sample 2130 can be sequentially exposed to only one type of ion ata time, or the exposure of sample 2130 can include exposing sample 2130to one or more ion beams that include two or more different types ofions. Different types of ions produce different ion dose profiles in anexposed material, and by exposing a sample to different types of ions, aparticular net ion dose profile in the sample can be realized. FIG. 62Ashows a schematic diagram of three different ion dose profiles 2010 g-ithat result from exposing a sample 2130 to three different types ofions. Ion dose profiles 2010 g-i can be produced via sequential exposureof the sample to each one of the different types of ions, or viaconcurrent exposure of the sample to two or even all three of thedifferent types of ions. The net ion dose profile in sample 2130 thatresults from exposure to the three different types of ions is shown inFIG. 62B. Variations in the net ion dose profile as a function ofthickness of the sample are reduced relative to any one of theindividual ion dose profiles shown in FIG. 62A.

In some embodiments, the different types of ions can include ions ofdifferent atomic composition. For example, the different types of ionscan include protons, carbon ions, oxygen ions, hydride ions, nitrogenions, chlorine ions, fluorine ions, argon ions, neon ions, krypton ions,and various types of metal ions such as sodium ions, calcium ions, andlithium ions. Generally, any of these different types of ions can beused to treat sample 2130, and each will produce a different ion doseprofile in a sample. In certain embodiments, ions can be generated fromcommonly available gases such as air. When air is used as a source gas,many different types of ions can be generated. The various differenttypes of ions can be separated from one another prior to exposing sample2130, or sample 2130 can be exposed to multiple different types of ionsgenerated from a source gas such as air.

In some embodiments, the different types of ions can include ions havingdifferent charges. For example, the different types of ions can includevarious positive and/or negative ions. Further, the different types ofions can include ions having single and/or multiple charges. In general,positive and negative ions of the same chemical species can producedifferent ion dose profiles in a particular sample, and ions of the samechemical species that have different charge magnitudes (e.g.,singly-charged, doubly-charged, triply-charged) can produce differention dose profiles in a particular sample. By exposing a sample tomultiple different types of ions, sample breakdown (e.g.,depolymerization, chain scission, and/or molecular weight reduction) canbe carefully and selectively controlled.

In some embodiments, the ion beam exposure system can adjust thecomposition of the ion beam based on the sample material. For example,certain types of sample, such as cellulosic biomass, include a largeconcentration of hydroxyl groups. Accordingly, the effective penetrationdepth of certain types of ions—particularly protons—in such materialscan be considerably larger than would other wise be expected based onion energy alone. Site-to-site proton hopping and other similar atomicexcursions can significantly increase the mobility of such ions in thesample, effectively increasing the penetration depth of the incidentions. Further, the increased mobility of the ions in the sample can leadto a broadening of the Bragg peak. The ion beam exposure system can beconfigured to select particular types of ions for exposure of certainsamples, accounting for the chemical and structural features of thesample. Further, the ion beam exposure system can be configured to takeinto account the expected interactions between the ion beam and thematerial when determining how to modify other parameters of the ion beamsuch as the distribution of ion energies therein.

The various techniques disclosed herein that are based on ion beamexposure of a biomass material can be used cooperatively with otherdisclosed techniques such as sonication, electron beam irradiation,chemical methods, and biological methods. The ion beam techniquesprovide significant advantages, including the ability to perform ionbeam exposure of dry samples, to deliver large radiation doses tosamples in short periods of time for high throughput applications, andto exercise relatively precise control over exposure conditions.

6. Ion-Beam Treatment of Biomass

A wide variety of different methods and systems can be used to produceion beams for treating biomass. In addition, ion beams produced usingthe systems and methods disclosed herein can be used alone to treatbiomass, or the ion beams can be used in combination with othertreatment methods (e.g., electron beams, sonication, biological agents,chemical treatments) to process biomass material.

An important aspect of the ion beam systems and methods disclosed hereinis that the disclosed systems and methods enable exposure of biomass toions in the presence of one or more additional fluids (e.g., gasesand/or liquids). Typically, for example, when a material is exposed toan ion beam, the exposure occurs in a reduced pressure environment suchas a vacuum chamber. The reduced pressure environment is used to reduceor prevent contamination of the exposed material, and also to reduce orprevent scattering of the ion beam by gas molecules. Unfortunately, ionbeam exposure of materials in closed environments such as a vacuumchamber greatly restricts potential throughput for high volume materialprocessing, however.

In the systems and methods disclosed herein, it has been recognized thatexposure of biomass to an ion beam in the presence of one or moreadditional fluids can increase the efficiency of the biomass treatment.Additionally, exposure of biomass to an ion beam in an open environment(e.g., in air at normal atmospheric pressure) provides for much higherthroughput than would otherwise be possible in a reduced pressureenvironment.

As discussed above, in some embodiments, biomass is exposed to an ionbeam in the presence of a fluid such as air. Ions accelerated in any oneor more of the types of accelerators disclosed herein (or another typeof accelerator) are coupled out of the accelerator via an output port(e.g., a thin membrane such as a metal foil), pass through a volume ofspace occupied by the fluid, and are then incident on the biomassmaterial. In addition to directly treating the biomass, some of the ionsgenerate additional chemical species by interacting with fluid particles(e.g., ions and/or radicals generated from various constituents of air).These generated chemical species can also interact with the biomass, andcan act as initiators for a variety of different chemical bond-breakingreactions in the biomass (e.g., depolymerization reactions).

In certain embodiments, additional fluids can be selectively introducedinto the path of an ion beam before the ion beam is incident on thebiomass. As discussed above, reactions between the ions and theparticles of the introduced fluids can generate additional chemicalspecies which react with the biomass and can assist in reducing themolecular weight of the biomass, and/or otherwise selectively alteringcertain properties of the biomass. The one or more additional fluids canbe directed into the path of the ion beam from a supply tube, forexample. The direction and flow rate of the fluid(s) that is/areintroduced can be selected according to a desired exposure rate and/ordirection to control the efficiency of the overall biomass treatment,including effects that result from both ion-based treatment and effectsthat are due to the interaction of dynamically generated species fromthe introduced fluid with the biomass. In addition to air, exemplaryfluids that can be introduced into the ion beam include oxygen,nitrogen, one or more noble gases, one or more halogens, and hydrogen.

In some embodiments, ion beams that include more that one different typeof ions can be used to treat biomass. Beams that include multipledifferent types of ions can be generated by combining two or moredifferent beams, each formed of one type of ion. Alternatively, or inaddition, in certain embodiments, ion beams that include multipledifferent types of ions can be generated by introducing a multicomponentsupply gas into and ion source and/or accelerator. For example, amulticomponent gas such as air can be used to generate an ion beamhaving different types of ions, including nitrogen ions, oxygen ions,argon ions, carbon ions, and other types of ions. Other multicomponentmaterials (e.g., gases, liquids, and solids) can be used to generate ionbeams having different compositions. Filtering elements (e.g.,hemispherical electrostatic filters) can be used to filter out certainionic constituents and/or neutral species to selectively produce an ionbeam having a particular composition, which can then be used to treatbiomass. By using air as a source for producing ion beams for biomasstreatment, the operating costs of a treatment system can be reducedrelative to systems that rely on pure materials, for example.

Certain types of biomass materials may be particularly amenable totreatment with multiple different types of ions and/or multipledifferent processing methods. For example, cellulosic materialstypically include crystalline polymeric cellulose chains which arecross-linked by amorphous hemicellulose fraction. The cellulose andhemicellulose is embedded within an amorphous lignin matrix. Separationof the cellulose fraction from the lignin and the hemicellulose usingconventional methods is difficult and can be energy-intensive.

However, cellulosic biomass can be treated with multiple different typesof ions to break down and separate the various components therein forfurther processing. In particular, the chemical properties of varioustypes of ionic species can be used to process cellulosic biomass (andother types of biomass) to selectively degrade and separate thecomponents thereof. For example, positively charged ions—and inparticular, protons—act as acids when exposed to biomass material.Conversely, negatively charged ions, particularly hydride ions, act asbases when exposed to biomass material. As a result, the chemicalproperties of these species can be used to target specific components oftreated biomass.

When treating lignocellulosic biomass, for example, the lignin matrixtypically decomposes in the presence of basic reagents. Accordingly, byfirst treating cellulosic biomass with basic ions such as hydride ions(or electrons) from an ion (electron) beam, the lignin fraction can bepreferentially degraded and separated from the cellulose andhemicellulose fractions. Cellulose is relatively unaffected by such anion treatment, as cellulose is typically stable in the presence of basicagents.

In addition to negative ion treatment (or as an alternative to negativeion treatment), the lignocellulosic biomass can be treated with one ormore basic agents in solution to assist in separating the lignin. Forexample, treatment of the lignocellulosic biomass with a sodiumbicarbonate solution can degrade and/or solubilize the lignin, enablingseparation of the solvated and/or suspended lignin from the celluloseand hemicellulose fractions.

Negative ion treatment with an ion beam may also assist in separatinghemicellulose, which is also chemically sensitive to basic reagents.Depending upon the particular structure of the cellulosic biomass, morethan treatment with negative ions may be used (and/or may be necessary)to effectively separate the hemicellulose fraction from the cellulosefraction. In addition, more that one type of ion can be used to separatethe hemicellulose. For example, a relatively less basic ion beam such asan oxygen ion beam can be used to treat cellulosic biomass to degradeand/or remove the lignin fraction. Then, a stronger basic ion beam suchas a hydride ion beam can be used to degrade and separate thehemicellulose from the cellulose. The cellulosic fraction remainslargely unchanged as a result of exposure to two different types ofbasic ions.

However, the cellulose fraction decomposes in the presence of acidicagents. Accordingly, a further processing step can include exposing thecellulose fraction to one or more acidic ions such as protons from anion beam, to assist in depolymerizing and/or degrading the cellulosefraction.

Each of the above ion treatments can be used in combination with otherprocessing steps. For example, separation steps (including introducing asolvent such as water) can be used to wash away particular fractions ofthe cellulosic biomass as they are degraded. Additional chemical agentscan be added to assist in separating the various components. Forexample, it has been observed that lignin that is separated from thecellulose and hemicellulose fractions can be suspended in a washingsolution. However, the lignin can readily re-deposit from the solutiononto the cellulose and hemicellulose fractions. To avoid re-depositionof the lignin, the suspension can be gently heated to ensure that thelignin remains below its glass transition temperature, and thereforeremains fluid. By maintaining the lignin below its glass transitiontemperature, the lignin can be more readily washed out of cellulosicbiomass. In general, heating of the suspension is carefully controlledto avoid thermal degradation of the sugars in the cellulosic fraction.

In addition, other treatment steps can be used to remove lignin fromcellulose and hemicellulose. For example, in certain embodiments,lignocellulosic biomass can first be treated with relatively heavy ions(e.g., carbon ions, oxygen ions) to degrade lignin, and the celluloseand hemicellulose can then be treated with relatively light ions (e.g.,protons, helium ions) and/or electrons to cause degradation of thecellulose and/or hemicellulose.

In some embodiments, one or more functionalizing agents can be added tothe suspension containing the lignin to enhance the solubility of ligninin solution, thereby discouraging re-deposition on the cellulose andhemicellulose fractions. For example, agents such as ammonia gas and/orvarious types of alcohols can be used (to introduce amino andhydroxyl/alkoxy groups, respectively) to functionalize the lignin.

In certain embodiments, structural agents can be added to the ligninsuspension to prevent re-deposition of the lignin onto the cellulose andhemicellulose fractions. Typically, when lignin forms a matrixsurrounding cellulose and/or hemicellulose, the lignin adopts a heavilyfolded structure, which permits relatively extensive van der Waalsinteractions with cellulose and hemicellulose. In contrast, when ligninis separated from cellulose and hemicellulose, the lignin adopts a moreopen, unfolded structure. By adding one or more agents that assist inpreventing lignin re-folding to the lignin suspension, re-association ofthe lignin with cellulose and hemicellulose can be discouraged, and thelignin can be more effectively removed via washing, for example.

Electromagnetic Radiation

In embodiments in which the irradiating is performed withelectromagnetic radiation, the electromagnetic radiation can have, e.g.,energy per photon (in electron volts) of greater than 10² eV, e.g.,greater than 10³, 10⁴, 10⁵, 10⁶, or even greater than 10⁷ eV. In someembodiments, the electromagnetic radiation has energy per photon ofbetween 10⁴ and 10⁷, e.g., between 10⁵ and 10⁶ eV. The electromagneticradiation can have a frequency of, e.g., greater than 10¹⁶ Hz, greaterthan 10¹⁷ Hz, 10¹⁸, 10¹⁹, 10²⁰, or even greater than 10²¹ Hz. In someembodiments, the electromagnetic radiation has a frequency of between10¹⁸ and 10²² Hz, e.g., between 10¹⁹ to 10²¹ Hz.

Doses

In some embodiments, the irradiating (with any radiation source or acombination of sources) is performed until the material receives a doseof at least 0.25 Mrad, e.g., at least 1.0 Mrad, at least 2.5 Mrad, atleast 5.0 Mrad, or at least 10.0 Mrad. In some embodiments, theirradiating is performed until the material receives a dose of between1.0 Mrad and 6.0 Mrad, e.g., between 1.5 Mrad and 4.0 Mrad.

In some embodiments, the irradiating is performed at a dose rate ofbetween 5.0 and 1500.0 kilorads/hour, e.g., between 10.0 and 750.0kilorads/hour or between 50.0 and 350.0 kilorads/hours.

In some embodiments, two or more radiation sources are used, such as twoor more ionizing radiations. For example, samples can be treated, in anyorder, with a beam of electrons, followed by gamma radiation and UVlight having wavelengths from about 100 nm to about 280 nm. In someembodiments, samples are treated with three ionizing radiation sources,such as a beam of electrons, gamma radiation, and energetic UV light.

In some embodiments, relatively low doses of radiation can crosslink,graft, or otherwise increase the molecular weight of acarbohydrate-containing material, such as a cellulosic orlignocellulosic material (e.g., cellulose). Such a material havingincreased molecular weight can be useful, e.g., in making a composite,e.g., having improved mechanical properties, such as abrasionresistance, compression strength, fracture resistance, impact strength,bending strength, tensile modulus, flexural modulus and elongation atbreak. Such a material having increased molecular weight can be usefulin making a composition.

For example, a fibrous material that includes a first cellulosic and/orlignocellulosic material having a first molecular weight can beirradiated in such a manner as to provide a second cellulosic and/orlignocellulosic material having a second molecular weight higher thanthe first molecular weight. For example, if gamma radiation is utilizedas the radiation source, a dose of from about 1 Mrad to about 10 Mrad,e.g., from about 1.5 Mrad to about 7.5 Mrad or from about 2.0 Mrad toabout 5.0 Mrad, can be applied. After the low dose of radiation, thesecond cellulosic and/or lignocellulosic material can be combined with aresin and formed into a composite, e.g., by compression molding,injection molding or extrusion. Forming composites is described in WO2006/102543, and in U.S. Provisional Patent Application Ser. No.60/664,832, filed on Mar. 24, 2005, 60/688,002, filed on Jun. 7, 2005,60/711,057, filed on Aug. 24, 2005, 60/715,822, filed on Sep. 9, 2005,60/725,674, filed on Oct. 12, 2005, 60/726,102, filed on Oct. 12, 2005,and 60/750,205, filed on Dec. 13, 2005.

Alternatively, a material, e.g., a fibrous material that includes afirst cellulosic and/or lignocellulosic material having a firstmolecular weight can be combined with a resin to provide a composite,and then the composite can be irradiated with a relatively low dose ofradiation so as to provide a second cellulosic and/or lignocellulosicmaterial having a second molecular weight higher than the firstmolecular weight. For example, if gamma radiation is utilized as theradiation source, a dose of from about 1 Mrad to about 10 Mrad can beapplied. Using this approach increases the molecular weight of thematerial while it is within a resin matrix. In some embodiments, theresin is a cross-linkable resin and as such it crosslinks as thecarbohydrate-containing material increases in molecular weight, whichcan provide a synergistic effect to provide advantageous mechanicalproperties to the composite. For example, such composites can haveexcellent low temperature performance, e.g., having a reduced tendencyto break and/or crack at low temperatures, e.g., temperatures below 0°C., e.g., below −10° C., −20° C., −40° C., −50° C., −60° C. or evenbelow −100° C., and/or excellent performance at high temperatures, e.g.,capable of maintaining their advantageous mechanical properties atrelatively high temperature, e.g., at temperatures above 100° C., e.g.,above 125° C., 150° C., 200° C., 250° C., 300° C., 400° C., or evenabove 500° C. In addition, such composites can have excellent chemicalresistance, e.g., resistance to swelling in a solvent, e.g., ahydrocarbon solvent, resistance to chemical attack, e.g., by strongacids, strong bases, strong oxidants (e.g., chlorine or bleach) orreducing agents (e.g., active metals such as sodium and potassium).

Alternatively, in another example, a fibrous material that includes acellulosic and/or lignocellulosic material is irradiated and,optionally, treated with acoustic energy, e.g., ultrasound.

In one example of the use of radiation as a pretreatment, half-gallonjuice cartons made of un-printed polycoated white Kraft board having abulk density of 20 lb/ft³ are used as a feedstock. Cartons are foldedflat and then fed into a sequence of three shredder-shearer trainsarranged in series with output from the first shearer fed as input tothe second shredder, and output from the second shearer fed as input tothe third shredder. The fibrous material produced by the can be sprayedwith water and processed through a pellet mill operating at roomtemperature. The densified pellets can be placed in a glass ampoulewhich is evacuated under high vacuum and then back-filled with argongas. The ampoule is sealed under argon. The pellets in the ampoule areirradiated with gamma radiation for about 3 hours at a dose rate ofabout 1 Mrad per hour to provide an irradiated material in which thecellulose has a lower molecular weight than the starting material.

Quenching and Controlled Functionalization of Biomass

After treatment with one or more ionizing radiations, such as photonicradiation (e.g., X-rays or gamma-rays), e-beam radiation or particlesheavier than electrons that are positively or negatively charged (e.g.,protons or carbon ions), any of the carbohydrate-containing materials ormixtures described herein become ionized; that is, they include radicalsat levels that are detectable with an electron spin resonancespectrometer. The current practical limit of detection of the radicalsis about 10¹⁴ spins at room temperature. After ionization, any biomassmaterial that has been ionized can be to reduce the level of radicals inthe ionized biomass, e.g., such that the radicals are no longerdetectable with the electron spin resonance spectrometer. For example,the radicals can be quenched by the application of a sufficient pressureto the biomass and/or utilizing a fluid in contact with the ionizedbiomass, such as a gas or liquid, that reacts with (quenches) theradicals. The use of a gas or liquid to at least aid in the quenching ofthe radicals also allows the operator to control functionalization ofthe ionized biomass with a desired amount and kinds of functionalgroups, such as carboxylic acid groups, enol groups, aldehyde groups,nitro groups, nitrile groups, amino groups, alkyl amino groups, alkylgroups, chloroalkyl groups or chlorofluoroalkyl groups. In someinstances, such quenching can improve the stability of some of theionized biomass materials. For example, quenching can improve thebiomass's resistance to oxidation. Functionalization by quenching canalso improve the solubility of any biomass described herein, can improveits thermal stability, which can be important in the manufacture ofcomposites and boards described herein, and can improve materialutilization by various microorganisms. For example, the functionalgroups imparted to the biomass material by quenching can act as receptorsites for attachment by microorganisms, e.g., to enhance cellulosehydrolysis by various microorganisms.

FIG. 11B illustrates changing a molecular and/or a supramolecularstructure of a biomass feedstock by pretreating the biomass feedstockwith ionizing radiation, such as with electrons or ions of sufficientenergy to ionize the biomass feedstock, to provide a first level ofradicals. As shown in FIG. 11B, if the ionized biomass remains in theatmosphere, it will be oxidized, such as to an extent that carboxylicacid groups are generated by reacting with the atmospheric oxygen. Insome instances with some materials, such oxidation is desired because itcan aid in the further breakdown in molecular weight of thecarbohydrate-containing biomass, and the oxidation groups, e.g.,carboxylic acid groups can be helpful for solubility and microorganismutilization in some instances. However, since the radicals can “live”for some time after irradiation, e.g., longer than 1 day, 5 days, 30days, 3 months, 6 months or even longer than 1 year, material propertiescan continue to change over time, which in some instances, can beundesirable. Detecting radicals in irradiated samples by electron spinresonance spectroscopy and radical lifetimes in such samples isdiscussed in Bartolotta et al., Physics in Medicine and Biology, 46(2001), 461-471 and in Bartolotta et al., Radiation ProtectionDosimetry, Vol. 84, Nos. 1-4, pp. 293-296 (1999), the contents of eachof which are incorporated herein by reference. As shown in FIG. 11B, theionized biomass can be quenched to functionalize and/or to stabilize theionized biomass. At any point, e.g., when the material is “alive”,“partially alive” or fully quenched, the pretreated biomass can beconverted into a product, e.g., a fuel, a food, or a composite.

In some embodiments, the quenching includes an application of pressureto the biomass, such as by mechanically deforming the biomass, e.g.,directly mechanically compressing the biomass in one, two, or threedimensions, or applying pressure to a fluid in which the biomass isimmersed, e.g., isostatic pressing. In such instances, the deformationof the material itself brings radicals, which are often trapped incrystalline domains, in sufficient proximity so that the radicals canrecombine, or react with another group. In some instances, the pressureis applied together with the application of heat, such as a sufficientquantity of heat to elevate the temperature of the biomass to above amelting point or softening point of a component of the biomass, such aslignin, cellulose or hemicellulose. Heat can improve molecular mobilityin the polymeric material, which can aid in the quenching of theradicals. When pressure is utilized to quench, the pressure can begreater than about 1000 psi, such as greater than about 1250 psi, 1450psi, 3625 psi, 5075 psi, 7250 psi, 10000 psi or even greater than 15000psi.

In some embodiments, quenching includes contacting the biomass with afluid, such as a liquid or gas, e.g., a gas capable of reacting with theradicals, such as acetylene or a mixture of acetylene in nitrogen,ethylene, chlorinated ethylenes or chlorofluoroethylenes, propylene ormixtures of these gases. In other particular embodiments, quenchingincludes contacting the biomass with a liquid, e.g., a liquid solublein, or at least capable of penetrating into the biomass and reactingwith the radicals, such as a diene, such as 1,5-cyclooctadiene. In somespecific embodiments, the quenching includes contacting the biomass withan antioxidant, such as Vitamin E. If desired, the biomass feedstock caninclude an antioxidant dispersed therein, and the quenching can comefrom contacting the antioxidant dispersed in the biomass feedstock withthe radicals.

Other methods for quenching are possible. For example, any method forquenching radicals in polymeric materials described in Muratoglu et al.,U.S. Patent Application Publication No. 2008/0067724 and Muratoglu etal., U.S. Pat. No. 7,166,650, the contents of each of which areincorporated herein by reference, can be utilized for quenching anyionized biomass material described herein. Furthermore any quenchingagent (described as a “sensitizing agent” in the above-noted Muratogludisclosures) and/or any antioxidant described in either Muratoglureference can be utilized to quench any ionized biomass material.

Functionalization can be enhanced by utilizing heavy charged ions, suchas any of the heavier ions described herein. For example, if it isdesired to enhance oxidation, charged oxygen ions can be utilized forthe irradiation. If nitrogen functional groups are desired, nitrogenions or an ions that includes nitrogen can be utilized. Likewise, ifsulfur or phosphorus groups are desired, sulfur or phosphorus ions canbe used in the irradiation.

In some embodiments, after quenching any of the quenched materialsdescribed herein can be further treated with one or more of radiation,such as ionizing or non-ionizing radiation, sonication, pyrolysis, andoxidation for additional molecular and/or supramolecular structurechange.

In particular embodiments, functionalized materials described herein aretreated with an acid, base, nucleophile or Lewis acid for additionalmolecular and/or supramolecular structure change, such as additionalmolecular weight breakdown. Examples of acids include organic acids,such as acetic acid and mineral acids, such as hydrochloric, sulfuricand/or nitric acid. Examples of bases include strong mineral bases, suchas a source of hydroxide ion, basic ions, such as fluoride ion, orweaker organic bases, such as amines. Even water and sodium bicarbonate,e.g., when dissolved in water, can effect molecular and/orsupramolecular structure change, such as additional molecular weightbreakdown.

Sonication

One or more sonication processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences. Sonication can reduce the molecular weight and/orcrystallinity of feedstock and biomass, e.g., one or more carbohydratesources, such as cellulosic or lignocellulosic materials, or starchymaterials.

Referring again to FIG. 8, in one method, a first material 2 thatincludes cellulose having a first number average molecular weight(^(T)M_(N1)) is dispersed in a medium, such as water, and sonicatedand/or otherwise cavitated, to provide a second material 3 that includescellulose having a second number average molecular weight (^(T)M_(N2))lower than the first number average molecular weight. The secondmaterial (or the first and second material in certain embodiments) canbe combined with a microorganism (e.g., a bacterium or a yeast) that canutilize the second and/or first material to produce a fuel 5 that is orincludes hydrogen, an alcohol, an organic acid, a hydrocarbon, ormixtures of any of these.

Since the second material has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable, and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 10⁶microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic, and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Sonicationcan also sterilize the materials, but should not be used while themicroorganisms are supposed to be alive.

In some embodiments, the second number average molecular weight(^(T)M_(N2)) is lower than the first number average molecular weight(^(T)M_(N1)) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, 50 percent, 60 percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has ascrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity index (prior tosonication) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after sonication is from about 10 to about 50percent, e.g., from about 15 to about 45 percent or from about 20 toabout 40 percent. However, in certain embodiments, e.g., after extensivesonication, it is possible to have a crystallinity index of lower than 5percent. In some embodiments, the material after sonication issubstantially amorphous.

In some embodiments, the starting number average molecular weight (priorto sonication) is from about 200,000 to about 3,200,000, e.g., fromabout 250,000 to about 1,000,000 or from about 250,000 to about 700,000,and the number average molecular weight after sonication is from about50,000 to about 200,000, e.g., from about 60,000 to about 150,000 orfrom about 70,000 to about 125,000. However, in some embodiments, e.g.,after extensive sonication, it is possible to have a number averagemolecular weight of less than about 10,000 or even less than about5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability, and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or microbial attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the sonication isperformed in an oxidizing medium, producing a second material that ismore oxidized than the first material. For example, the second materialcan have more hydroxyl groups, aldehyde groups, ketone groups, estergroups or carboxylic acid groups, which can increase its hydrophilicity.

In some embodiments, the sonication medium is an aqueous medium. Ifdesired, the medium can include an oxidant, such as a peroxide (e.g.,hydrogen peroxide), a dispersing agent and/or a buffer. Examples ofdispersing agents include ionic dispersing agents, e.g., sodium laurylsulfate, and non-ionic dispersing agents, e.g., poly(ethylene glycol).

In other embodiments, the sonication medium is non-aqueous. For example,the sonication can be performed in a hydrocarbon, e.g., toluene orheptane, an ether, e.g., diethyl ether or tetrahydrofuran, or even in aliquefied gas such as argon, xenon, or nitrogen.

Without wishing to be bound by any particular theory, it is believedthat sonication breaks bonds in the cellulose by creating bubbles in themedium containing the cellulose, which grow and then violently collapse.During the collapse of the bubble, which can take place in less than ananosecond, the implosive force raises the local temperature within thebubble to about 5100 K (even higher in some instance; see, e.g., Suslicket al., Nature 434, 52-55, 2005) and generates pressures of from a fewhundred atmospheres to over 1000 atmospheres or more. It is these hightemperatures and pressures that break the bonds. In addition, withoutwishing to be bound by any particular theory, it is believed thatreduced crystallinity arises, at least in part, from the extremely highcooling rates during collapse of the bubbles, which can be greater thanabout 10¹¹ K/second. The high cooling rates generally do not allow thecellulose to organize and crystallize, resulting in materials that havereduced crystallinity. Ultrasonic systems and sonochemistry arediscussed in, e.g., Olli et al., U.S. Pat. No. 5,766,764; Roberts, U.S.Pat. No. 5,828,156; Mason, Chemistry with Ultrasound, Elsevier, Oxford,(1990); Suslick (editor), Ultrasound: its Chemical, Physical andBiological Effects, VCH, Weinheim, (1988); Price, “Current Trends inSonochemistry” Royal Society of Chemistry, Cambridge, (1992); Suslick etal., Ann. Rev. Mater. Sci. 29, 295, (1999); Suslick et al., Nature 353,414 (1991); Hiller et al., Phys. Rev. Lett. 69, 1182 (1992); Barber etal., Nature, 352, 414 (1991); Suslick et al., J. Am. Chem. Soc., 108,5641 (1986); Tang et al., Chem. Comm., 2119 (2000); Wang et al.,Advanced Mater., 12, 1137 (2000); Landau et al., J. of Catalysis, 201,22 (2001); Perkas et al., Chem. Comm., 988 (2001); Nikitenko et al.,Angew. Chem. Inter. Ed. (December 2001); Shafi et al., J. Phys. Chem B103, 3358 (1999); Avivi et al., J. Amer. Chem. Soc. 121, 4196 (1999);and Avivi et al., J. Amer. Chem. Soc. 122, 4331 (2000).

Sonication Systems

FIG. 12 shows a general system in which a cellulosic material stream1210 is mixed with a water stream 1212 in a reservoir 1214 to form aprocess stream 1216. A first pump 1218 draws process stream 1216 fromreservoir 1214 and toward a flow cell 1224. Ultrasonic transducer 1226transmits ultrasonic energy into process stream 1216 as the processstream flows through flow cell 1224. A second pump 1230 draws processstream 1216 from flow cell 1224 and toward subsequent processing.

Reservoir 1214 includes a first intake 1232 and a second intake 1234 influid communication with a volume 1236. A conveyor (not shown) deliverscellulosic material stream 1210 to reservoir 1214 through first intake1232. Water stream 1212 enters reservoir 1214 through second intake1234. In some embodiments, water stream 1212 enters volume 1236 along atangent establishing a swirling flow within volume 1236. In certainembodiments, cellulosic material stream 1210 and water stream 1212 areintroduced into volume 1236 along opposing axes to enhance mixing withinthe volume.

Valve 1238 controls the flow of water stream 1212 through second intake1232 to produce a desired ratio of cellulosic material to water (e.g.,approximately 10% cellulosic material, weight by volume). For example,2000 tons/day of cellulosic material can be combined with 1 million to1.5 million gallons/day, e.g., 1.25 million gallons/day, of water.

Mixing of cellulosic material and water in reservoir 1214 is controlledby the size of volume 1236 and the flow rates of cellulosic material andwater into the volume. In some embodiments, volume 1236 is sized tocreate a minimum mixing residence time for the cellulosic material andwater. For example, when 2000 tons/day of cellulosic material and 1.25million gallons/day of water are flowing through reservoir 1214, volume1236 can be about 32,000 gallons to produce a minimum mixing residencetime of about 15 minutes.

Reservoir 1214 includes a mixer 1240 in fluid communication with volume1236. Mixer 1240 agitates the contents of volume 1236 to dispersecellulosic material throughout the water in the volume. For example,mixer 1240 can be a rotating vane disposed in reservoir 1214. In someembodiments, mixer 1240 disperses the cellulosic material substantiallyuniformly throughout the water.

Reservoir 1214 further includes an exit 1242 in fluid communication withvolume 1236 and process stream 1216. The mixture of cellulosic materialand water in volume 1236 flows out of reservoir 1214 via exit 1242. Exit1242 is arranged near the bottom of reservoir 1214 to allow gravity topull the mixture of cellulosic material and water out of reservoir 1214and into process stream 1216.

First pump 1218 (e.g., any of several recessed impeller vortex pumpsmade by Essco Pumps & Controls, Los Angeles, Calif.) moves the contentsof process stream 1216 toward flow cell 1224. In some embodiments, firstpump 1218 agitates the contents of process stream 1216 such that themixture of cellulosic material and water is substantially uniform atinlet 1220 of flow cell 1224. For example, first pump 1218 agitatesprocess stream 1216 to create a turbulent flow that persists along theprocess stream between the first pump and inlet 1220 of flow cell 1224.

Flow cell 1224 includes a reactor volume 1244 in fluid communicationwith inlet 1220 and outlet 1222. In some embodiments, reactor volume1244 is a stainless steel tube capable of withstanding elevatedpressures (e.g., 10 bars). In addition or in the alternative, reactorvolume 1244 includes a rectangular cross section.

Flow cell 1224 further includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 is sonicated in reactor volume 1244.In some embodiments, the flow rate of cooling fluid 1248 into heatexchanger 1246 is controlled to maintain an approximately constanttemperature in reactor volume 1244. In addition or in the alternative,the temperature of cooling fluid 1248 flowing into heat exchanger 1246is controlled to maintain an approximately constant temperature inreactor volume 1244. In some embodiments, the temperature of reactorvolume 1244 is maintained at 20 to 50° C., e.g., 25, 30, 35, 40, or 45°C. Additionally or alternatively, heat transferred to cooling fluid 1248from reactor volume 1244 can be used in other parts of the overallprocess.

An adapter section 1226 creates fluid communication between reactorvolume 1244 and a booster 1250 coupled (e.g., mechanically coupled usinga flange) to ultrasonic transducer 1226. For example, adapter section1226 can include a flange and O-ring assembly arranged to create a leaktight connection between reactor volume 1244 and booster 1250. In someembodiments, ultrasonic transducer 1226 is a high-powered ultrasonictransducer made by Hielscher Ultrasonics of Teltow, Germany.

In operation, a generator 1252 delivers electricity to ultrasonictransducer 1252. Ultrasonic transducer 1226 includes a piezoelectricelement that converts the electrical energy into sound in the ultrasonicrange. In some embodiments, the materials are sonicated using soundhaving a frequency of from about 16 kHz to about 110 kHz, e.g., fromabout 18 kHz to about 75 kHz or from about 20 kHz to about 40 kHz.(e.g., sound having a frequency of 20 kHz to 40 kHz).

The ultrasonic energy is then delivered to the working medium throughbooster 1248.

The ultrasonic energy traveling through booster 1248 in reactor volume1244 creates a series of compressions and rarefactions in process stream1216 with an intensity sufficient to create cavitation in process stream1216. Cavitation disaggregates the cellulosic material dispersed inprocess stream 1216. Cavitation also produces free radicals in the waterof process stream 1216. These free radicals act to further break downthe cellulosic material in process stream 1216.

In general, 5 to 4000 MJ/m³, e.g., 10, 25, 50, 100, 250, 500, 750, 1000,2000, or 3000 MJ/m³, of ultrasonic energy is applied to process stream16 flowing at a rate of about 0.2 m³/s (about 3200 gallons/min). Afterexposure to ultrasonic energy in reactor volume 1244, process stream1216 exits flow cell 1224 through outlet 1222. Second pump 1230 movesprocess stream 1216 to subsequent processing (e.g., any of severalrecessed impeller vortex pumps made by Essco Pumps & Controls, LosAngeles, Calif.).

While certain embodiments have been described, other embodiments arepossible.

As an example, while process stream 1216 has been described as a singleflow path, other arrangements are possible. In some embodiments forexample, process stream 1216 includes multiple parallel flow paths(e.g., flowing at a rate of 10 gallon/min). In addition or in thealternative, the multiple parallel flow paths of process stream 1216flow into separate flow cells and are sonicated in parallel (e.g., usinga plurality of 16 kW ultrasonic transducers).

As another example, while a single ultrasonic transducer 1226 has beendescribed as being coupled to flow cell 1224, other arrangements arepossible. In some embodiments, a plurality of ultrasonic transducers1226 are arranged in flow cell 1224 (e.g., ten ultrasonic transducerscan be arranged in a flow cell 1224). In some embodiments, the soundwaves generated by each of the plurality of ultrasonic transducers 1226are timed (e.g., synchronized out of phase with one another) to enhancethe cavitation acting upon process stream 1216.

As another example, while a single flow cell 1224 has been described,other arrangements are possible. In some embodiments, second pump 1230moves process stream to a second flow cell where a second booster andultrasonic transducer further sonicate process stream 1216.

As still another example, while reactor volume 1244 has been describedas a closed volume, reactor volume 1244 is open to ambient conditions incertain embodiments. In such embodiments, sonication pretreatment can beperformed substantially simultaneously with other pretreatmenttechniques. For example, ultrasonic energy can be applied to processstream 1216 in reactor volume 1244 while electron beams aresimultaneously introduced into process stream 1216.

As another example, while a flow through process has been described,other arrangements are possible. In some embodiments, sonication can beperformed in a batch process. For example, a volume can be filled with a10% (weight by volume) mixture of cellulosic material in water andexposed to sound with intensity from about 50 W/cm² to about 600 W/cm²,e.g., from about 75 W/cm² to about 300 W/cm² or from about 95 W/cm² toabout 200 W/cm². Additionally or alternatively, the mixture in thevolume can be sonicated from about 1 hour to about 24 hours, e.g., fromabout 1.5 hours to about 12 hours, or from about 2 hours to about 10hours. In certain embodiments, the material is sonicated for apre-determined time, and then allowed to stand for a secondpre-determined time before sonicating again.

Referring now to FIG. 13, in some embodiments, two electro-acoustictransducers are mechanically coupled to a single horn. As shown, a pairof piezoelectric transducers 60 and 62 is coupled to a slotted bar horn64 by respective intermediate coupling horns 70 and 72, the latter alsobeing known as booster horns. The mechanical vibrations provided by thetransducers, responsive to high frequency electrical energy appliedthereto, are transmitted to the respective coupling horns, which may beconstructed to provide a mechanical gain, such as a ratio of 1 to 1.2.The horns are provided with a respective mounting flange 74 and 76 forsupporting the transducer and horn assembly in a stationary housing.

The vibrations transmitted from the transducers through the coupling orbooster horns are coupled to the input surface 78 of the horn and aretransmitted through the horn to the oppositely disposed output surface80, which, during operation, is in forced engagement with a workpiece(not shown) to which the vibrations are applied.

The high frequency electrical energy provided by the power supply 82 isfed to each of the transducers, electrically connected in parallel, viaa balancing transformer 84 and a respective series connected capacitor86 and 90, one capacitor connected in series with the electricalconnection to each of the transducers. The balancing transformer isknown also as “balun” standing for “balancing unit.” The balancingtransformer includes a magnetic core 92 and a pair of identical windings94 and 96, also termed the primary winding and secondary winding,respectively.

In some embodiments, the transducers include commercially availablepiezoelectric transducers, such as Branson Ultrasonics Corporationmodels 105 or 502, each designed for operation at 20 kHz and a maximumpower rating of 3 kW. The energizing voltage for providing maximummotional excursion at the output surface of the transducer is 930 voltrms. The current flow through a transducer may vary between zero and 3.5ampere depending on the load impedance. At 930 volt rms the outputmotion is approximately 20 microns. The maximum difference in terminalvoltage for the same motional amplitude, therefore, can be 186 volt.Such a voltage difference can give rise to large circulating currentsflowing between the transducers. The balancing unit 430 assures abalanced condition by providing equal current flow through thetransducers, hence eliminating the possibility of circulating currents.The wire size of the windings must be selected for the full load currentnoted above and the maximum voltage appearing across a winding input is93 volt.

As an alternative to using ultrasonic energy, high-frequency,rotor-stator devices can be utilized. This type of device produceshigh-shear, microcavitation forces which can disintegrate biomass incontact with such forces. Two commercially available high-frequency,rotor-stator dispersion devices are the SUPRATRON™ devices manufacturedby Krupp Industrietechnik GmbH and marketed by Dorr-Oliver DeutschlandGmbH of Connecticut, and the DISPAX™ devices manufactured and marketedby Ika-Works, Inc. of Cincinnati, Ohio. Operation of such amicrocavitation device is discussed in Stuart, U.S. Pat. No. 5,370,999.

While ultrasonic transducer 1226 has been described as including one ormore piezoelectric active elements to create ultrasonic energy, otherarrangements are possible. In some embodiments, ultrasonic transducer1226 includes active elements made of other types of magnetostrictivematerials (e.g., ferrous metals). Design and operation of such ahigh-powered ultrasonic transducer is discussed in Hansen et al., U.S.Pat. No. 6,624,539. In some embodiments, ultrasonic energy istransferred to process stream 16 through an electro-hydraulic system.

While ultrasonic transducer 1226 has been described as using theelectromagnetic response of magnetorestrictive materials to produceultrasonic energy, other arrangements are possible. In some embodiments,acoustic energy in the form of an intense shock wave can be applieddirectly to process stream 16 using an underwater spark. In someembodiments, ultrasonic energy is transferred to process stream 16through a thermo-hydraulic system. For example, acoustic waves of highenergy density can be produced by applying power across an enclosedvolume of electrolyte, thereby heating the enclosed volume and producinga pressure rise that is subsequently transmitted through a soundpropagation medium (e.g., process stream 1216). Design and operation ofsuch a thermo-hydraulic transducer is discussed in Hartmann et al., U.S.Pat. No. 6,383,152.

Pyrolysis

One or more pyrolysis processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences.

Referring again to the general schematic in FIG. 8, a first material 2that includes cellulose having a first number average molecular weight(^(T)M_(N1)) is pyrolyzed, e.g., by heating the first material in a tubefurnace, to provide a second material 3 that includes cellulose having asecond number average molecular weight (^(T)M_(N2)) lower than the firstnumber average molecular weight. The second material (or the first andsecond material in certain embodiments) is/are combined with amicroorganism (e.g., a bacterium or a yeast) that can utilize the secondand/or first material to produce a fuel 5 that is or includes hydrogen,an alcohol (e.g., ethanol or butanol, such as n-, sec or t-butanol), anorganic acid, a hydrocarbon or mixtures of any of these.

Since the second material has cellulose having a reduced molecularweight relative to the first material, and in some instances, a reducedcrystallinity as well, the second material is generally moredispersible, swellable and/or soluble in a solution containing themicroorganism, e.g., at a concentration of greater than 10⁶microorganisms/mL. These properties make the second material 3 moresusceptible to chemical, enzymatic and/or microbial attack relative tothe first material 2, which can greatly improve the production rateand/or production level of a desired product, e.g., ethanol. Pyrolysiscan also sterilize the first and second materials.

In some embodiments, the second number average molecular weight(^(T)M_(N2)) is lower than the first number average molecular weight(^(T)M_(N1)) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, 50 percent, 60 percent, or even more than about 75 percent.

In some instances, the second material has cellulose that has ascrystallinity (^(T)C₂) that is lower than the crystallinity (^(T)C₁) ofthe cellulose of the first material. For example, (^(T)C₂) can be lowerthan (^(T)C₁) by more than about 10 percent, e.g., 15, 20, 25, 30, 35,40, or even more than about 50 percent.

In some embodiments, the starting crystallinity (prior to pyrolysis) isfrom about 40 to about 87.5 percent, e.g., from about 50 to about 75percent or from about 60 to about 70 percent, and the crystallinityindex after pyrolysis is from about 10 to about 50 percent, e.g., fromabout 15 to about 45 percent or from about 20 to about 40 percent.However, in certain embodiments, e.g., after extensive pyrolysis, it ispossible to have a crystallinity index of lower than 5 percent. In someembodiments, the material after pyrolysis is substantially amorphous.

In some embodiments, the starting number average molecular weight (priorto pyrolysis) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after pyrolysis is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive pyrolysis, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second material can have a level of oxidation(^(T)O₂) that is higher than the level of oxidation (^(T)O₁) of thefirst material. A higher level of oxidation of the material can aid inits dispersibility, swellability and/or solubility, further enhancingthe materials susceptibility to chemical, enzymatic or microbial attack.In some embodiments, to increase the level of the oxidation of thesecond material relative to the first material, the pyrolysis isperformed in an oxidizing environment, producing a second material thatis more oxidized than the first material. For example, the secondmaterial can have more hydroxyl groups, aldehyde groups, ketone groups,ester groups or carboxylic acid groups, which can increase itshydrophilicity.

In some embodiments, the pyrolysis of the materials is continuous. Inother embodiments, the material is pyrolyzed for a pre-determined time,and then allowed to cool for a second pre-determined time beforepyrolyzing again.

Pyrolysis Systems

FIG. 14 shows a process flow diagram 6000 that includes various steps ina pyrolytic feedstock pretreatment system. In first step 6010, a supplyof dry feedstock is received from a feed source.

As described above, the dry feedstock from the feed source may bepre-processed prior to delivery to the pyrolysis chamber. For example,if the feedstock is derived from plant sources, certain portions of theplant material may be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing 6020 (e.g., to reduce the averagelength of fibers in the feedstock) prior to delivery to the pyrolysischamber.

Following mechanical processing, the feedstock undergoes a moistureadjustment step 6030. The nature of the moisture adjustment step dependsupon the moisture content of the mechanically processed feedstock.Typically, pyrolysis of feedstock occurs most efficiently when themoisture content of the feedstock is between about 10% and about 30%(e.g., between 15% and 25%) by weight of the feedstock. If the moisturecontent of the feedstock is larger than about 40% by weight, the extrathermal load presented by the water content of the feedstock increasesthe energy consumption of subsequent pyrolysis steps.

In some embodiments, if the feedstock has a moisture content which islarger than about 30% by weight, drier feedstock material 6220 which hasa low moisture content can be blended in, creating a feedstock mixturein step 6030 with an average moisture content that is within the limitsdiscussed above. In certain embodiments, feedstock with a high moisturecontent can simply be dried by dispersing the feedstock material on amoving conveyor that cycles the feedstock through an in-line heatingunit. The heating unit evaporates a portion of the water present in thefeedstock.

In some embodiments, if the feedstock from step 6020 has a moisturecontent which is too low (e.g., lower than about 10% by weight), themechanically processed feedstock can be combined with wetter feedstockmaterial 6230 with a higher moisture content, such as sewage sludge.Alternatively, or in addition, water 6240 can be added to the dryfeedstock from step 6020 to increase its moisture content.

In step 6040, the feedstock—now with its moisture content adjusted tofall within suitable limits—can be preheated in an optional preheatingstep 6040. Preheating step 6040 can be used to increase the temperatureof the feedstock to between 75° C. and 150° C. in preparation forsubsequent pyrolysis of the feedstock. Depending upon the nature of thefeedstock and the particular design of the pyrolysis chamber, preheatingthe feedstock can ensure that heat distribution within the feedstockremains more uniform during pyrolysis, and can reduce the thermal loadon the pyrolysis chamber.

The feedstock is then transported to a pyrolysis chamber to undergopyrolysis in step 6050. In some embodiments, transport of the feedstockis assisted by adding one or more pressurized gases 6210 to thefeedstock stream. The gases create a pressure gradient in a feedstocktransport conduit, propelling the feedstock into the pyrolysis chamber(and even through the pyrolysis chamber). In certain embodiments,transport of the feedstock occurs mechanically; that is, a transportsystem that includes a conveyor such as an auger transports thefeedstock to the pyrolysis chamber.

Other gases 6210 can also be added to the feedstock prior to thepyrolysis chamber. In some embodiments, for example, one or morecatalyst gases can be added to the feedstock to assist decomposition ofthe feedstock during pyrolysis. In certain embodiments, one or morescavenging agents can be added to the feedstock to trap volatilematerials released during pyrolysis. For example, various sulfur-basedcompounds such as sulfides can be liberated during pyrolysis, and anagent such as hydrogen gas can be added to the feedstock to causedesulfurization of the pyrolysis products. Hydrogen combines withsulfides to form hydrogen sulfide gas, which can be removed from thepyrolyzed feedstock.

Pyrolysis of the feedstock within the chamber can include heating thefeedstock to relatively high temperatures to cause partial decompositionof the feedstock. Typically, the feedstock is heated to a temperature ina range from 150° C. to 1100° C. The temperature to which the feedstockis heated depends upon a number of factors, including the composition ofthe feedstock, the feedstock average particle size, the moisturecontent, and the desired pyrolysis products. For many types of biomassfeedstock, for example, pyrolysis temperatures between 300° C. and 550°C. are used.

The residence time of the feedstock within the pyrolysis chambergenerally depends upon a number of factors, including the pyrolysistemperature, the composition of the feedstock, the feedstock averageparticle size, the moisture content, and the desired pyrolysis products.In some embodiments, feedstock materials are pyrolyzed at a temperaturejust above the decomposition temperature for the material in an inertatmosphere, e.g., from about 2° C. above to about 10° C. above thedecomposition temperature or from about 3° C. above to about 7° C. abovethe decomposition temperature. In such embodiments, the material isgenerally kept at this temperature for greater than 0.5 hour, e.g.,greater than 1.0 hour or greater than about 2.0 hours. In otherembodiments, the materials are pyrolyzed at a temperature well above thedecomposition temperature for the material in an inert atmosphere, e.g.,from about 75° C. above to about 175° C. above the decompositiontemperature or from about 85° C. above to about 150° C. above thedecomposition temperature. In such embodiments, the material isgenerally kept at this temperature for less than 0.5 hour, e.g., less 20minutes, less than 10 minutes, less than 5 minutes or less than 2minutes. In still other embodiments, the materials are pyrolyzed at anextreme temperature, e.g., from about 200° C. above to about 500° C.above the decomposition temperature of the material in an inertenvironment or from about 250° C. above to about 400° C. above thedecomposition temperature. In such embodiments, the material usgenerally kept at this temperature for less than 1 minute, e.g., lessthan 30 seconds, less than 15 seconds, less than 10 seconds, less than 5seconds, less than 1 second or less than 500 ms. Such embodiments aretypically referred to as flash pyrolysis.

In some embodiments, the feedstock is heated relatively rapidly to theselected pyrolysis temperature within the chamber. For example, thechamber can be designed to heat the feedstock at a rate of between 500°C./s and 11,000° C./s. Typical heating rates for biomass-derivedfeedstock material are from 500° C./s to 1000° C./s, for example.

A turbulent flow of feedstock material within the pyrolysis chamber isusually advantageous, as it ensures relatively efficient heat transferto the feedstock material from the heating sub-system. Turbulent flowcan be achieved by blowing the feedstock material through the chamberusing one or more injected carrier gases 6210, for example. In general,the carrier gases are relatively inert towards the feedstock material,even at the high temperatures in the pyrolysis chamber. Exemplarycarrier gases include, for example, nitrogen, argon, methane, carbonmonoxide, and carbon dioxide. Alternatively, or in addition, mechanicaltransport systems such as augers can transport and circulate thefeedstock within the pyrolysis chamber to create a turbulent feedstockflow.

In some embodiments, pyrolysis of the feedstock occurs substantially inthe absence of oxygen and other reactive gases. Oxygen can be removedfrom the pyrolysis chamber by periodic purging of the chamber with highpressure nitrogen (e.g., at nitrogen pressures of 2 bar or more).Following purging of the chamber, a gas mixture present in the pyrolysischamber (e.g., during pyrolysis of the feedstock) can include less than4 mole % oxygen (e.g., less than 1 mole % oxygen, and even less than 0.5mole % oxygen). The absence of oxygen ensures that ignition of thefeedstock does not occur at the elevated pyrolysis temperatures.

In certain embodiments, relatively small amounts of oxygen can beintroduced into the feedstock and are present during pyrolysis. Thistechnique is referred to as oxidative pyrolysis. Typically, oxidativepyrolysis occurs in multiple heating stages. For example, in a firstheating stage, the feedstock is heated in the presence of oxygen tocause partial oxidation of the feedstock. This stage consumes theavailable oxygen in the pyrolysis chamber. Then, in subsequent heatingstages, the feedstock temperature is further elevated. With all of theoxygen in the chamber consumed, however, feedstock combustion does notoccur, and combustion-free pyrolytic decomposition of the feedstock(e.g., to generate hydrocarbon products) occurs. In general, the processof heating feedstock in the pyrolysis chamber to initiate decompositionis endothermic. However, in oxidative pyrolysis, formation of carbondioxide by oxidation of the feedstock is an exothermic process. The heatreleased from carbon dioxide formation can assist further pyrolysisheating stages, thereby lessening the thermal load presented by thefeedstock.

In some embodiments, pyrolysis occurs in an inert environment, such aswhile feedstock materials are bathed in argon or nitrogen gas. Incertain embodiments, pyrolysis can occur in an oxidizing environment,such as in air or argon enriched in air. In some embodiments, pyrolysiscan take place in a reducing environment, such as while feedstockmaterials are bathed in hydrogen gas. To aid pyrolysis, various chemicalagents, such as oxidants, reductants, acids or bases can be added to thematerial prior to or during pyrolysis. For example, sulfuric acid can beadded, or a peroxide (e.g., benzoyl peroxide) can be added.

As discussed above, a variety of different processing conditions can beused, depending upon factors such as the feedstock composition and thedesired pyrolysis products. For example, for cellulose-containingfeedstock material, relatively mild pyrolysis conditions can beemployed, including flash pyrolysis temperatures between 375° C. and450° C., and residence times of less than 1 second. As another example,for organic solid waste material such as sewage sludge, flash pyrolysistemperatures between 500° C. and 650° C. are typically used, withresidence times of between 0.5 and 3 seconds. In general, many of thepyrolysis process parameters, including residence time, pyrolysistemperature, feedstock turbulence, moisture content, feedstockcomposition, pyrolysis product composition, and additive gas compositioncan be regulated automatically by a system of regulators and anautomated control system.

Following pyrolysis step 6050, the pyrolysis products undergo aquenching step 6250 to reduce the temperature of the products prior tofurther processing. Typically, quenching step 6250 includes spraying thepyrolysis products with streams of cooling water 6260. The cooling wateralso forms a slurry that includes solid, undissolved product materialand various dissolved products. Also present in the product stream is amixture that includes various gases, including product gases, carriergases, and other types of process gases.

The product stream is transported via in-line piping to a gas separatorthat performs a gas separation step 6060, in which product gases andother gases are separated from the slurry formed by quenching thepyrolysis products. The separated gas mixture is optionally directed toa blower 6130, which increases the gas pressure by blowing air into themixture. The gas mixture can be subjected to a filtration step 6140, inwhich the gas mixture passes through one or more filters (e.g.,activated charcoal filters) to remove particulates and other impurities.In a subsequent step 6150, the filtered gas can be compressed and storedfor further use. Alternatively, the filtered gas can be subjected tofurther processing steps 6160. For example, in some embodiments, thefiltered gas can be condensed to separate different gaseous compoundswithin the gas mixture. The different compounds can include, forexample, various hydrocarbon products (e.g., alcohols, alkanes, alkenes,alkynes, ethers) produced during pyrolysis. In certain embodiments, thefiltered gas containing a mixture of hydrocarbon components can becombined with steam gas 6170 (e.g., a mixture of water vapor and oxygen)and subjected to a cracking process to reduce molecular weights of thehydrocarbon components.

In some embodiments, the pyrolysis chamber includes heat sources thatburn hydrocarbon gases such as methane, propane, and/or butane to heatthe feedstock. A portion 6270 of the separated gases can be recirculatedinto the pyrolysis chamber for combustion, to generate process heat tosustain the pyrolysis process.

In certain embodiments, the pyrolysis chamber can receive process heatthat can be used to increase the temperature of feedstock materials. Forexample, irradiating feedstock with radiation (e.g., gamma radiation,electron beam radiation, or other types of radiation) can heat thefeedstock materials to relatively high temperatures. The heatedfeedstock materials can be cooled by a heat exchange system that removessome of the excess heat from the irradiated feedstock. The heat exchangesystem can be configured to transport some of the heat energy to thepyrolysis chamber to heat (or pre-heat) feedstock material, therebyreducing energy cost for the pyrolysis process.

The slurry containing liquid and solid pyrolysis products can undergo anoptional de-watering step 6070, in which excess water can be removedfrom the slurry via processes such as mechanical pressing andevaporation. The excess water 6280 can be filtered and then recirculatedfor further use in quenching the pyrolysis decomposition products instep 6250.

The de-watered slurry then undergoes a mechanical separation step 6080,in which solid product material 6110 is separated from liquid productmaterial 6090 by a series of increasingly-fine filters. In step 6100,the liquid product material 6090 can then be condensed (e.g., viaevaporation) to remove waste water 6190, and purified by processes suchas extraction. Extraction can include the addition of one or moreorganic solvents 6180, for example, to separate products such as oilsfrom products such as alcohols. Suitable organic solvents include, forexample, various hydrocarbons and halo-hydrocarbons. The purified liquidproducts 6200 can then be subjected to further processing steps. Wastewater 6190 can be filtered if necessary, and recirculated for furtheruse in quenching the pyrolysis decomposition products in step 6250.

After separation in step 6080, the solid product material 6110 isoptionally subjected to a drying step 6120 that can include evaporationof water. Solid material 6110 can then be stored for later use, orsubjected to further processing steps, as appropriate.

The pyrolysis process parameters discussed above are exemplary. Ingeneral, values of these parameters can vary widely according to thenature of the feedstock and the desired products. Moreover, a widevariety of different pyrolysis techniques, including using heat sourcessuch as hydrocarbon flames and/or furnaces, infrared lasers, microwaveheaters, induction heaters, resistive heaters, and other heating devicesand configurations can be used.

A wide variety of different pyrolysis chambers can be used to decomposethe feedstock. In some embodiments, for example, pyrolyzing feedstockcan include heating the material using a resistive heating member, suchas a metal filament or metal ribbon. The heating can occur by directcontact between the resistive heating member and the material.

In certain embodiments, pyrolyzing can include heating the material byinduction, such as by using a Currie-Point pyrolyzer. In someembodiments, pyrolyzing can include heating the material by theapplication of radiation, such as infrared radiation. The radiation canbe generated by a laser, such as an infrared laser.

In certain embodiments, pyrolyzing can include heating the material witha convective heat. The convective heat can be generated by a flowingstream of heated gas. The heated gas can be maintained at a temperatureof less than about 1200° C., such as less than 1000° C., less than 750°C., less than 600° C., less than 400° C. or even less than 300° C. Theheated gas can be maintained at a temperature of greater than about 250°C. The convective heat can be generated by a hot body surrounding thefirst material, such as in a furnace.

In some embodiments, pyrolyzing can include heating the material withsteam at a temperature above about 250° C.

An embodiment of a pyrolysis chamber is shown in FIG. 15. Chamber 6500includes an insulated chamber wall 6510 with a vent 6600 for exhaustgases, a plurality of burners 6520 that generate heat for the pyrolysisprocess, a transport duct 6530 for transporting the feedstock throughchamber 6500, augers 6590 for moving the feedstock through duct 6530 ina turbulent flow, and a quenching system 6540 that includes an auger6610 for moving the pyrolysis products, water jets 6550 for spraying thepyrolysis products with cooling water, and a gas separator forseparating gaseous products 6580 from a slurry 6570 containing solid andliquid products.

Another embodiment of a pyrolysis chamber is shown in FIG. 16. Chamber6700 includes an insulated chamber wall 6710, a feedstock supply duct6720, a sloped inner chamber wall 6730, burners 6740 that generate heatfor the pyrolysis process, a vent 6750 for exhaust gases, and a gasseparator 6760 for separating gaseous products 6770 from liquid andsolid products 6780. Chamber 6700 is configured to rotate in thedirection shown by arrow 6790 to ensure adequate mixing and turbulentflow of the feedstock within the chamber.

A further embodiment of a pyrolysis chamber is shown in FIG. 17.Filament pyrolyzer 1712 includes a sample holder 1713 with resistiveheating element 1714 in the form of a wire winding through the openspace defined by the sample holder 1713. Optionally, the heated elementcan be spun about axis 1715 (as indicated by arrow 1716) to tumble thematerial that includes the cellulosic material in sample holder 1713.The space 1718 defined by enclosure 1719 is maintained at a temperatureabove room temperature, e.g., 200 to 250° C. In a typical usage, acarrier gas, e.g., an inert gas, or an oxidizing or reducing gas,traverses through the sample holder 1713 while the resistive heatingelement is rotated and heated to a desired temperature, e.g., 325° C.After an appropriate time, e.g., 5 to 10 minutes, the pyrolyzed materialis emptied from the sample holder. The system shown in FIG. 17 can bescaled and made continuous. For example, rather than a wire as theheating member, the heating member can be an auger screw. Material cancontinuously fall into the sample holder, striking a heated screw thatpyrolizes the material. At the same time, the screw can push thepyrolyzed material out of the sample holder to allow for the entry offresh, unpyrolyzed material.

Another embodiment of a pyrolysis chamber is shown in FIG. 18, whichfeatures a Curie-Point pyrolyzer 1820 that includes a sample chamber1821 housing a ferromagnetic foil 1822. Surrounding the sample chamber1821 is an RF coil 1823. The space 1824 defined by enclosure 1825 ismaintained at a temperature above room temperature, e.g., 200 to 250° C.In a typical usage, a carrier gas traverses through the sample chamber1821 while the foil 1822 is inductively heated by an applied RF field topyrolize the material at a desired temperature.

Yet another embodiment of a pyrolysis chamber is shown in FIG. 19.Furnace pyrolyzer 130 includes a movable sample holder 131 and a furnace132. In a typical usage, the sample is lowered (as indicated by arrow137) into a hot zone 135 of furnace 132, while a carrier gas fills thehousing 136 and traverses through the sample holder 131. The sample isheated to the desired temperature for a desired time to provide apyrolyzed product. The pyrolyzed product is removed from the pyrolyzerby raising the sample holder (as indicated by arrow 134).

In certain embodiments, as shown in FIG. 20, a cellulosic target 140 canbe pyrolyzed by treating the target, which is housed in a vacuum chamber141, with laser light, e.g., light having a wavelength of from about 225nm to about 1500 nm. For example, the target can be ablated at 266 nm,using the fourth harmonic of a Nd-YAG laser (Spectra Physics, GCR170,San Jose, Calif.). The optical configuration shown allows the nearlymonochromatic light 143 generated by the laser 142 to be directed usingmirrors 144 and 145 onto the target after passing though a lens 146 inthe vacuum chamber 141. Typically, the pressure in the vacuum chamber ismaintained at less than about 10⁻⁶ mm Hg. In some embodiments, infraredradiation is used, e.g., 1.06 micron radiation from a Nd-YAG laser. Insuch embodiments, a infrared sensitive dye can be combined with thecellulosic material to produce a cellulosic target. The infrared dye canenhance the heating of the cellulosic material. Laser ablation isdescribed by Blanchet-Fincher et al. in U.S. Pat. No. 5,942,649.

Referring to FIG. 21, in some embodiments, a cellulosic material can beflash pyrolyzed by coating a tungsten filament 150, such as a 5 to 25mil tungsten filament, with the desired cellulosic material while thematerial is housed in a vacuum chamber 151. To affect pyrolysis, currentis passed through the filament, which causes a rapid heating of thefilament for a desired time. Typically, the heating is continued forseconds before allowing the filament to cool. In some embodiments, theheating is performed a number of times to effect the desired amount ofpyrolysis.

In certain embodiments, carbohydrate-containing biomass material can beheated in an absence of oxygen in a fluidized bed reactor. If desired,the carbohydrate containing biomass can have relatively thincross-sections, and can include any of the fibrous materials describedherein, for efficient heat transfer. The material can be heated bythermal transfer from a hot metal or ceramic, such as glass beads orsand in the reactor, and the resulting pyrolysis liquid or oil can betransported to a central refinery for making combustible fuels or otheruseful products.

Oxidation

One or more oxidative processing sequences can be used to process rawfeedstock from a wide variety of different sources to extract usefulsubstances from the feedstock, and to provide partially degraded organicmaterial which functions as input to further processing steps and/orsequences.

Referring again to FIG. 8, a first material 2 that includes cellulosehaving a first number average molecular weight (^(T)M_(N1)) and having afirst oxygen content (^(T)O₁) is oxidized, e.g., by heating the firstmaterial in a tube furnace in stream of air or oxygen-enriched air, toprovide a second material 3 that includes cellulose having a secondnumber average molecular weight (^(T)M_(N2)) and having a second oxygencontent (^(T)O₂) higher than the first oxygen content (^(T)O₁). Thesecond material (or the first and second material in certainembodiments) can be, e.g., combined with a resin, such as a moltenthermoplastic resin or a microorganism, to provide a composite 4 havingdesirable mechanical properties, or a fuel 5. Providing a higher levelof oxidation can improve dispersibility of the oxidized material in aresin and can also improve the interfacial bond between the oxidizedmaterial and the resin. Improved dispersibility and/or interfacialbonding (in some instances in combination with maintaining molecularweight) can provide composites with exceptional mechanical properties,such as improved abrasion resistance, compression strength, fractureresistance, impact strength, bending strength, tensile modulus, flexuralmodulus and elongation at break.

Such materials can also be combined with a solid and/or a liquid. Forexample, the liquid can be in the form of a solution and the solid canbe particulate in form. The liquid and/or solid can include amicroorganism, e.g., a bacterium, and/or an enzyme. For example, thebacterium and/or enzyme can work on the cellulosic or lignocellulosicmaterial to produce a fuel, such as ethanol, or a coproduct, such as aprotein. Exemplary fuels and coproducts are described in FIBROUSMATERIALS AND COMPOSITES,” U.S. Ser. No. 11/453,951, filed Jun. 15,2006.

In some embodiments, the second number average molecular weight is notmore 97 percent lower than the first number average molecular weight,e.g., not more than 95 percent, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45,40, 30, 20, 12.5, 10.0, 7.5, 5.0, 4.0, 3.0, 2.5, 2.0 or not more than1.0 percent lower than the first number average molecular weight. Theamount of reduction of molecular weight will depend upon theapplication. For example, in some preferred embodiments that providecomposites, the second number average molecular weight is substantiallythe same as the first number average molecular weight. In otherapplications, such as making ethanol or another fuel or coproduct, ahigher amount of molecular weight reduction is generally preferred.

For example, in some embodiments that provide a composite, the startingnumber average molecular weight (prior to oxidation) is from about200,000 to about 3,200,000, e.g., from about 250,000 to about 1,000,000or from about 250,000 to about 700,000, and the number average molecularweight after oxidation is from about 175,000 to about 3,000,000, e.g.,from about 200,000 to about 750,000 or from about 225,000 to about600,000.

Resins utilized can be thermosets or thermoplastics. Examples ofthermoplastic resins include rigid and elastomeric thermoplastics. Rigidthermoplastics include polyolefins (e.g., polyethylene, polypropylene,or polyolefin copolymers), polyesters (e.g., polyethyleneterephthalate), polyamides (e.g., nylon 6, 6/12 or 6/10), andpolyethyleneimines. Examples of elastomeric thermoplastic resins includeelastomeric styrenic copolymers (e.g., styrene-ethylene-butylene-styrenecopolymers), polyamide elastomers (e.g., polyether-polyamide copolymers)and ethylene-vinyl acetate copolymer.

In particular embodiments, lignin is utilized, e.g., any lignin that isgenerated in any process described herein.

In some embodiments, the thermoplastic resin has a melt flow rate ofbetween 10 g/10 minutes to 60 g/10 minutes, e.g., between 20 g/10minutes to 50 g/10 minutes, or between 30 g/10 minutes to 45 g/10minutes, as measured using ASTM 1238. In certain embodiments, compatibleblends of any of the above thermoplastic resins can be used.

In some embodiments, the thermoplastic resin has a polydispersity index(PDI), i.e., a ratio of the weight average molecular weight to thenumber average molecular weight, of greater than 1.5, e.g., greater than2.0, greater than 2.5, greater than 5.0, greater than 7.5, or evengreater than 10.0.

In specific embodiments, polyolefins or blends of polyolefins areutilized as the thermoplastic resin.

Examples of thermosetting resins include natural rubber,butadiene-rubber and polyurethanes.

In some embodiments in which the materials are used to make a fuel or acoproduct, the starting number average molecular weight (prior tooxidation) is from about 200,000 to about 3,200,000, e.g., from about250,000 to about 1,000,000 or from about 250,000 to about 700,000, andthe number average molecular weight after oxidation is from about 50,000to about 200,000, e.g., from about 60,000 to about 150,000 or from about70,000 to about 125,000. However, in some embodiments, e.g., afterextensive oxidation, it is possible to have a number average molecularweight of less than about 10,000 or even less than about 5,000.

In some embodiments, the second oxygen content is at least about fivepercent higher than the first oxygen content, e.g., 7.5 percent higher,10.0 percent higher, 12.5 percent higher, 15.0 percent higher or 17.5percent higher. In some preferred embodiments, the second oxygen contentis at least about 20.0 percent higher than the oxygen content of thefirst material. Oxygen content is measured by elemental analysis bypyrolyzing a sample in a furnace operating 1300° C. or higher. Asuitable elemental analyzer is the LECO CHNS-932 analyzer with a VTF-900high temperature pyrolysis furnace.

In some embodiments, oxidation of first material 200 does not result ina substantial change in the crystallinity of the cellulose. However, insome instances, e.g., after extreme oxidation, the second material hascellulose that has as crystallinity (^(T)C₂) that is lower than thecrystallinity (^(T)C₁) of the cellulose of the first material. Forexample, (^(T)C₂) can be lower than (^(T)C₁) by more than about 5percent, e.g., 10, 15, 20, or even 25 percent. This can be desirablewhen optimizing the flexural fatigue properties of the composite is agoal. For example, reducing the crystallinity can improve the elongationat break or can enhance the impact resistance of a composite. This canalso be desirable to enhance solubility of the materials in a liquid,such as a liquid that includes a bacterium and/or an enzyme.

In some embodiments, the starting crystallinity index (prior tooxidation) is from about 40 to about 87.5 percent, e.g., from about 50to about 75 percent or from about 60 to about 70 percent, and thecrystallinity index after oxidation is from about 30 to about 75.0percent, e.g., from about 35.0 to about 70.0 percent or from about 37.5to about 65.0 percent. However, in certain embodiments, e.g., afterextensive oxidation, it is possible to have a crystallinity index oflower than 5 percent. In some embodiments, the material after oxidationis substantially amorphous.

Without wishing to be bound by any particular theory, it is believedthat oxidation increases the number of hydrogen-bonding groups on thecellulose, such as hydroxyl groups, aldehyde groups, ketone groupscarboxylic acid groups or anhydride groups, which can increase itsdispersibility and/or its solubility (e.g., in a liquid). To furtherimprove dispersibility in a resin, the resin can include a componentthat includes hydrogen-bonding groups, such as one or more anhydridegroups, carboxylic acid groups, hydroxyl groups, amide groups, aminegroups or mixtures of any of these groups. In some preferredembodiments, the component includes a polymer copolymerized with and/orgrafted with maleic anhydride. Such materials are available from DuPontunder the trade name FUSABOND®.

Generally, oxidation of first material 200 occurs in an oxidizingenvironment. For example, the oxidation can be effected or aided bypyrolysis in an oxidizing environment, such as in air or argon enrichedin air. To aid in the oxidation, various chemical agents, such asoxidants, acids or bases can be added to the material prior to or duringoxidation. For example, a peroxide (e.g., benzoyl peroxide) can be addedprior to oxidation.

Oxidation Systems

FIG. 22 shows a process flow diagram 5000 that includes various steps inan oxidative feedstock pretreatment system. In first step 5010, a supplyof dry feedstock is received from a feed source. The feed source caninclude, for example, a storage bed or container that is connected to anin-line oxidation reactor via a conveyor belt or another feedstocktransport device.

As described above, the dry feedstock from the feed source may bepre-processed prior to delivery to the oxidation reactor. For example,if the feedstock is derived from plant sources, certain portions of theplant material may be removed prior to collection of the plant materialand/or before the plant material is delivered by the feedstock transportdevice. Alternatively, or in addition, the biomass feedstock can besubjected to mechanical processing (e.g., to reduce the average lengthof fibers in the feedstock) prior to delivery to the oxidation reactor.

Following mechanical processing 5020, feedstock 5030 is transported to amixing system which introduces water 5150 into the feedstock in amechanical mixing process. Combining water with the processed feedstockin mixing step 5040 creates an aqueous feedstock slurry 5050, which canthen be treated with one or more oxidizing agents.

Typically, one liter of water is added to the mixture for every 0.02 kgto 1.0 kg of dry feedstock. The ratio of feedstock to water in themixture depends upon the source of the feedstock and the specificoxidizing agents used further downstream in the overall process. Forexample, in typical industrial processing sequences for lignocellulosicbiomass, aqueous feedstock slurry 5050 includes from about 0.5 kg toabout 1.0 kg of dry biomass per liter of water.

In some embodiments, one or more fiber-protecting additives 5170 canalso be added to the feedstock slurry in feedstock mixing step 5040.Fiber-protecting additives help to reduce degradation of certain typesof biomass fibers (e.g., cellulose fibers) during oxidation of thefeedstock. Fiber-protecting additives can be used, for example, if adesired product from processing a lignocellulosic feedstock includescellulose fibers. Exemplary fiber-protecting additives include magnesiumcompounds such as magnesium hydroxide. Concentrations offiber-protecting additives in feedstock slurry 5050 can be from 0.1% to0.4% of the dry weight of the biomass feedstock, for example.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional extraction 5180 with an organic solvent to removewater-insoluble substances from the slurry. For example, extraction ofslurry 5050 with one or more organic solvents yields a purified slurryand an organic waste stream 5210 that includes water-insoluble materialssuch as fats, oils, and other non-polar, hydrocarbon-based substances.Suitable solvents for performing extraction of slurry 5050 includevarious alcohols, hydrocarbons, and halo-hydrocarbons, for example.

In some embodiments, aqueous feedstock slurry 5050 can be subjected toan optional thermal treatment 5190 to further prepare the feedstock foroxidation. An example of a thermal treatment includes heating thefeedstock slurry in the presence of pressurized steam. In fibrousbiomass feedstock, the pressurized steam swells the fibers, exposing alarger fraction of fiber surfaces to the aqueous solvent and tooxidizing agents that are introduced in subsequent processing steps.

In certain embodiments, aqueous feedstock slurry 5050 can be subjectedto an optional treatment with basic agents 5200. Treatment with one ormore basic agents can help to separate lignin from cellulose inlignocellulosic biomass feedstock, thereby improving subsequentoxidation of the feedstock. Exemplary basic agents include alkali andalkaline earth hydroxides such as sodium hydroxide, potassium hydroxide,and calcium hydroxide. In general, a variety of basic agents can beused, typically in concentrations from about 0.01% to about 0.5% of thedry weight of the feedstock.

Aqueous feedstock slurry 5050 is transported (e.g., by an in-line pipingsystem) to a chamber, which can be an oxidation preprocessing chamber oran oxidation reactor. In oxidation preprocessing step 5060, one or moreoxidizing agents 5160 are added to feedstock slurry 5050 to form anoxidizing medium. In some embodiments, for example, oxidizing agents5160 can include hydrogen peroxide. Hydrogen peroxide can be added toslurry 5050 as an aqueous solution, and in proportions ranging from 3%to between 30% and 35% by weight of slurry 5050. Hydrogen peroxide has anumber of advantages as an oxidizing agent. For example, aqueoushydrogen peroxide solution is relatively inexpensive, is relativelychemically stable, and is not particularly hazardous relative to otheroxidizing agents (and therefore does not require burdensome handlingprocedures and expensive safety equipment). Moreover, hydrogen peroxidedecomposes to form water during oxidation of feedstock, so that wastestream cleanup is relatively straightforward and inexpensive.

In certain embodiments, oxidizing agents 5160 can include oxygen (e.g.,oxygen gas) either alone, or in combination with hydrogen peroxide.Oxygen gas can be bubbled into slurry 5050 in proportions ranging from0.5% to 10% by weight of slurry 5050. Alternatively, or in addition,oxygen gas can also be introduced into a gaseous phase in equilibriumwith slurry 5050 (e.g., a vapor head above slurry 5050). The oxygen gascan be introduced into either an oxidation preprocessing chamber or intoan oxidation reactor (or into both), depending upon the configuration ofthe oxidative processing system. Typically, for example, the partialpressure of oxygen in the vapor above slurry 5050 is larger than theambient pressure of oxygen, and ranges from 0.5 bar to 35 bar, dependingupon the nature of the feedstock.

The oxygen gas can be introduced in pure form, or can be mixed with oneor more carrier gases. For example, in some embodiments, high-pressureair provides the oxygen in the vapor. In certain embodiments, oxygen gascan be supplied continuously to the vapor phase to ensure that aconcentration of oxygen in the vapor remains within certainpredetermined limits during processing of the feedstock. In someembodiments, oxygen gas can be introduced initially in sufficientconcentration to oxidize the feedstock, and then the feedstock can betransported to a closed, pressurized vessel (e.g., an oxidation reactor)for processing.

In certain embodiments, oxidizing agents 5160 can include nascent oxygen(e.g., oxygen radicals). Typically, nascent oxygen is produced as neededin an oxidation reactor or in a chamber in fluid communication with anoxidation reactor by one or more decomposition reactions. For example,in some embodiments, nascent oxygen can be produced from a reactionbetween NO and O₂ in a gas mixture or in solution. In certainembodiments, nascent oxygen can be produced from decomposition of HOClin solution. Other methods by which nascent oxygen can be producedinclude via electrochemical generation in electrolyte solution, forexample.

In general, nascent oxygen is an efficient oxidizing agent due to therelatively high reactivity of the oxygen radical. However, nascentoxygen can also be a relatively selective oxidizing agent. For example,when lignocellulosic feedstock is treated with nascent oxygen, selectiveoxidation of lignin occurs in preference to the other components of thefeedstock such as cellulose. As a result, oxidation of feedstock withnascent oxygen provides a method for selective removal of the ligninfraction in certain feedstocks. Typically, nascent oxygen concentrationsof between about 0.5% and 5% of the dry weight of the feedstock are usedto effect efficient oxidation.

Without wishing to be bound by theory, it is believed that nascentoxygen reacts with lignocellulosic feedstock according to at least twodifferent mechanisms. In a first mechanism, nascent oxygen undergoes anaddition reaction with the lignin, resulting in partial oxidation of thelignin, which solubilizes the lignin in aqueous solution. As a result,the solubilized lignin can be removed from the rest of the feedstock viawashing. In a second mechanism, nascent oxygen disrupts butanecross-links and/or opens aromatic rings that are connected via thebutane cross-links. As a result, solubility of the lignin in aqueoussolution increases, and the lignin fraction can be separated from theremainder of the feedstock via washing.

In some embodiments, oxidizing agents 5160 include ozone (O₃). The useof ozone can introduce several chemical handling considerations in theoxidation processing sequence. If heated too vigorously, an aqueoussolution of ozone can decompose violently, with potentially adverseconsequences for both human system operators and system equipment.Accordingly, ozone is typically generated in a thermally isolated,thick-walled vessel separate from the vessel that contains the feedstockslurry, and transported thereto at the appropriate process stage.

Without wishing to be bound by theory, it is believed that ozonedecomposes into oxygen and oxygen radicals, and that the oxygen radicals(e.g., nascent oxygen) are responsible for the oxidizing properties ofozone in the manner discussed above. Ozone typically preferentiallyoxidizes the lignin fraction in lignocellulosic materials, leaving thecellulose fraction relatively undisturbed.

Conditions for ozone-based oxidation of biomass feedstock generallydepend upon the nature of the biomass. For example, for cellulosicand/or lignocellulosic feedstocks, ozone concentrations of from 0.1 g/m³to 20 g/m³ of dry feedstock provide for efficient feedstock oxidation.Typically, the water content in slurry 5050 is between 10% by weight and80% by weight (e.g., between 40% by weight and 60% by weight). Duringozone-based oxidation, the temperature of slurry 5050 can be maintainedbetween 0° C. and 100° C. to avoid violent decomposition of the ozone.

In some embodiments, feedstock slurry 5050 can be treated with anaqueous, alkaline solution that includes one or more alkali and alkalineearth hydroxides such as sodium hydroxide, potassium hydroxide, andcalcium hydroxide, and then treated thereafter with an ozone-containinggas in an oxidation reactor. This process has been observed tosignificantly increase decomposition of the biomass in slurry 5050.Typically, for example, a concentration of hydroxide ions in thealkaline solution is between 0.001% and 10% by weight of slurry 5050.After the feedstock has been wetted via contact with the alkalinesolution, the ozone-containing gas is introduced into the oxidationreactor, where it contacts and oxidizes the feedstock.

Oxidizing agents 5160 can also include other substances. In someembodiments, for example, halogen-based oxidizing agents such aschlorine and oxychlorine agents (e.g., hypochlorite) can be introducedinto slurry 5050. In certain embodiments, nitrogen-containing oxidizingsubstances can be introduced into slurry 5050. Exemplarynitrogen-containing oxidizing substances include NO and NO₂, forexample. Nitrogen-containing agents can also be combined with oxygen inslurry 5050 to create additional oxidizing agents. For example, NO andNO₂ both combine with oxygen in slurry 5050 to form nitrate compounds,which are effective oxidizing agents for biomass feedstock. Halogen- andnitrogen-based oxidizing agents can, in some embodiments, causebleaching of the biomass feedstock, depending upon the nature of thefeedstock. The bleaching may be desirable for certain biomass-derivedproducts that are extracted in subsequent processing steps.

Other oxidizing agents can include, for example, various peroxyacids,peroxyacetic acids, persulfates, percarbonates, permanganates, osmiumtetroxide, and chromium oxides.

Following oxidation preprocessing step 5060, feedstock slurry 5050 isoxidized in step 5070. If oxidizing agents 5160 were added to slurry5050 in an oxidation reactor, then oxidation proceeds in the samereactor. Alternatively, if oxidizing agents 5160 were added to slurry5050 in a preprocessing chamber, then slurry 5050 is transported to anoxidation reactor via an in-line piping system. Once inside theoxidation reactor, oxidation of the biomass feedstock proceeds under acontrolled set of environmental conditions. Typically, for example, theoxidation reactor is a cylindrical vessel that is closed to the externalenvironment and pressurized. Both batch and continuous operation ispossible, although environmental conditions are typically easier tocontrol in in-line batch processing operations.

Oxidation of feedstock slurry 5050 typically occurs at elevatedtemperatures in the oxidation reactor. For example, the temperature ofslurry 5050 in the oxidation reactor is typically maintained above 100°C., in a range from 120° C. to 240° C. For many types of biomassfeedstock, oxidation is particularly efficient if the temperature ofslurry 5050 is maintained between 150° C. and 220° C. Slurry 5050 can beheating using a variety of thermal transfer devices. For example, insome embodiments, the oxidation reactor contacts a heating bath thatincludes oil or molten salts. In certain embodiments, a series of heatexchange pipes surround and contact the oxidation reactor, andcirculation of hot fluid within the pipes heats slurry 5050 in thereactor. Other heating devices that can be used to heat slurry 5050include resistive heating elements, induction heaters, and microwavesources, for example.

The residence time of feedstock slurry 5050 in the oxidation reactor canbe varied as desired to process the feedstock. Typically, slurry 5050spends from 1 minute to 60 minutes undergoing oxidation in the reactor.For relatively soft biomass material such as lignocellulosic matter, theresidence time in the oxidation reactor can be from 5 minutes to 30minutes, for example, at an oxygen pressure of between 3 and 12 bars inthe reactor, and at a slurry temperature of between 160° C. and 210° C.For other types of feedstock, however, residence times in the oxidationreactor can be longer, e.g., as long 48 hours. To determine appropriateresidence times for slurry 5050 in the oxidation reactor, aliquots ofthe slurry can be extracted from the reactor at specific intervals andanalyzed to determine concentrations of particular products of interestsuch as complex saccharides. Information about the increase inconcentrations of certain products in slurry 5050 as a function of timecan be used to determine residence times for particular classes offeedstock material.

In some embodiments, during oxidation of feedstock slurry 5050,adjustment of the slurry pH may be performed by introducing one or morechemical agents into the oxidation reactor. For example, in certainembodiments, oxidation occurs most efficiently in a pH range of about9-11. To maintain a pH in this range, agents such as alkali and alkalineearth hydroxides, carbonates, ammonia, and alkaline buffer solutions canbe introduced into the oxidation reactor.

Circulation of slurry 5050 during oxidation can be important to ensuresufficient contact between oxidizing agents 5160 and the feedstock.Circulation of the slurry can be achieved using a variety of techniques.For example, in some embodiments, a mechanical stirring apparatus thatincludes impeller blades or a paddle wheel can be implemented in theoxidation reactor. In certain embodiments, the oxidation reactor can bea loop reactor, in which the aqueous solvent in which the feedstock issuspended is simultaneously drained from the bottom of the reactor andrecirculated into the top of the reactor via pumping, thereby ensuringthat the slurry is continually re-mixed and does not stagnate within thereactor.

After oxidation of the feedstock is complete, the slurry is transportedto a separation apparatus where a mechanical separation step 5080occurs. Typically, mechanical separation step 5080 includes one or morestages of increasingly-fine filtering of the slurry to mechanicallyseparate the solid and liquid constituents.

Liquid phase 5090 is separated from solid phase 5100, and the two phasesare processed independently thereafter. Solid phase 5100 can optionallyundergo a drying step 5120 in a drying apparatus, for example. Dryingstep 5120 can include, for example, mechanically dispersing the solidmaterial onto a drying surface, and evaporating water from solid phase5100 by gentle heating of the solid material. Following drying step 5120(or, alternatively, without undergoing drying step 5120), solid phase5100 is transported for further processing steps 5140.

Liquid phase 5090 can optionally undergo a drying step 5110 to reducethe concentration of water in the liquid phase. In some embodiments, forexample, drying step 5110 can include evaporation and/or distillationand/or extraction of water from liquid phase 5090 by gentle heating ofthe liquid. Alternatively, or in addition, one or more chemical dryingagents can be used to remove water from liquid phase 5090. Followingdrying step 5110 (or alternatively, without undergoing drying step5110), liquid phase 5090 is transported for further processing steps5130, which can include a variety of chemical and biological treatmentsteps such as chemical and/or enzymatic hydrolysis.

Drying step 5110 creates waste stream 5220, an aqueous solution that caninclude dissolved chemical agents such as acids and bases in relativelylow concentrations. Treatment of waste stream 5220 can include, forexample, pH neutralization with one or more mineral acids or bases.Depending upon the concentration of dissolved salts in waste stream5220, the solution may be partially de-ionized (e.g., by passing thewaste stream through an ion exchange system). Then, the wastestream—which includes primarily water—can be re-circulated into theoverall process (e.g., as water 5150), diverted to another process, ordischarged.

Typically, for lignocellulosic biomass feedstocks following separationstep 5070, liquid phase 5090 includes a variety of soluble poly- andoligosaccharides, which can then be separated and/or reduced tosmaller-chain saccharides via further processing steps. Solid phase 5100typically includes primarily cellulose, for example, with smalleramounts of hemicellulose- and lignin-derived products.

In some embodiments, oxidation can be carried out at elevatedtemperature in a reactor such as a pyrolysis chamber. For example,referring again to FIG. 17, feedstock materials can be oxidized infilament pyrolyzer 1712. In a typical usage, an oxidizing carrier gas,e.g., air or an air/argon blend, traverses through the sample holder1713 while the resistive heating element is rotated and heated to adesired temperature, e.g., 325° C. After an appropriate time, e.g., 5 to10 minutes, the oxidized material is emptied from the sample holder. Thesystem shown in FIG. 2 can be scaled and made continuous. For example,rather than a wire as the heating member, the heating member can be anauger screw. Material can continuously fall into the sample holder,striking a heated screw that pyrolizes the material. At the same time,the screw can push the oxidized material out of the sample holder toallow for the entry of fresh, unoxidized material.

Referring again to FIG. 18, feedstock materials can be oxidized in aCurie-Point pyrolyzer 1820. In a typical usage, an oxidizing carrier gastraverses through the sample chamber 1821 while the foil 1822 isinductively heated by an applied RF field to oxidize the material at adesired temperature.

Referring again to FIG. 19, feedstock materials can be oxidized in afurnace pyrolyzer 130. In a typical usage, the sample is lowered (asindicated by arrow 137) into a hot zone 135 of furnace 132, while anoxidizing carrier gas fills the housing 136 and traverses through thesample holder 131. The sample is heated to the desired temperature for adesired time to provide an oxidized product. The oxidized product isremoved from the pyrolyzer by raising the sample holder (as indicated byarrow 134).

Referring again to FIG. 20, feedstock materials can be oxidized byforming a cellulosic target 140, along with an oxidant, such as aperoxide, and treating the target, which is housed in a vacuum chamber141, with laser light, e.g., light having a wavelength of from about 225nm to about 1600 nm. The optical configuration shown allows themonochromatic light 143 generated by the laser 142 to be directed usingmirrors 144 and 145 onto the target after passing though a lens 146 inthe vacuum chamber 141. Typically, the pressure in the vacuum chamber ismaintained at less than about 10⁻⁶ mm Hg. In some embodiments, infraredradiation is used, e.g., 1.06 micron radiation from a Nd-YAG laser. Insuch embodiments, a infrared sensitive dye can be combined with thecellulosic material to produce a cellulosic target. The infrared dye canenhance the heating of the cellulosic material. Laser treatment ofpolymers is described by Blanchet-Fincher et al. in U.S. Pat. No.5,942,649.

Referring again to FIG. 21, feedstock materials can be rapidly oxidizedby coating a tungsten filament 150, together with an oxidant, such as aperoxide, with the desired cellulosic material while the material ishoused in a vacuum chamber 151. To affect oxidation, current is passedthrough the filament, which causes a rapid heating of the filament for adesired time. Typically, the heating is continued for seconds beforeallowing the filament to cool. In some embodiments, the heating isperformed a number of times to effect the desired amount of oxidation.

Referring again to FIG. 12, in some embodiments, feedstock materials canbe oxidized with the aid of sound and/or cavitation. Generally, toeffect oxidation, the materials are sonicated in an oxidizingenvironment, such as water saturated with oxygen or another chemicaloxidant, such as hydrogen peroxide.

Referring again to FIGS. 9 and 10, in certain embodiments, ionizingradiation is used to aid in the oxidation of feedstock materials.Generally, to effect oxidation, the materials are irradiated in anoxidizing environment, such as air or oxygen. For example, gammaradiation and/or electron beam radiation can be employed to irradiatethe materials.

Other Processes

Steam explosion can be used alone without any of the processes describedherein, or in combination with any of the processes described herein.

FIG. 23 shows an overview of the entire process of converting a fibersource 400 into a product 450, such as ethanol, by a process thatincludes shearing and steam explosion to produce a fibrous material 401,which is then hydrolyzed and converted, e.g., fermented, to produce theproduct. The fiber source can be transformed into the fibrous material401 through a number of possible methods, including at least oneshearing process and at least one steam explosion process.

For example, one option includes shearing the fiber source, followed byoptional screening step(s) and optional additional shearing step(s) toproduce a sheared fiber source 402, which can then be steam exploded toproduce the fibrous material 401. The steam explosion process isoptionally followed by a fiber recovery process to remove liquids or the“liquor” 404, resulting from the steam exploding process. The materialresulting from steam exploding the sheared fiber source may be furthersheared by optional additional shearing step(s) and/or optionalscreening step(s).

In another method, the fibrous material 401 is first steam exploded toproduce a steam exploded fiber source 410. The resulting steam explodedfiber source is then subjected to an optional fiber recovery process toremove liquids, or the liquor. The resulting steam exploded fiber sourcecan then be sheared to produce the fibrous material. The steam explodedfiber source can also be subject to one or more optional screening stepsand/or one or more optional additional shearing steps. The process ofshearing and steam exploding the fiber source to produce the sheared andsteam exploded fibrous material will be further discussed below.

The fiber source can be cut into pieces or strips of confetti materialprior to shearing or steam explosion. The shearing processes can takeplace in a dry (e.g., having less than 0.25 percent by weight absorbedwater), hydrated, or even while the material is partially or fullysubmerged in a liquid, such as water or isopropanol. The process canalso optimally include steps of drying the output after steam explodingor shearing to allow for additional steps of dry shearing or steamexploding. The steps of shearing, screening, and steam explosion cantake place with or without the presence of various chemical solutions.

In a steam explosion process, the fiber source or the sheared fibersource is contacted with steam under high pressure, and the steamdiffuses into the structures of the fiber source (e.g., thelignocellulosic structures). The steam then condenses under highpressure thereby “wetting” the fiber source. The moisture in the fibersource can hydrolyze any acetyl groups in the fiber source (e.g., theacetyl groups in the hemicellulose fractions), forming organic acidssuch as acetic and uronic acids. The acids, in turn, can catalyze thedepolymerization of hemicellulose, releasing xylan and limited amountsof glucan. The “wet” fiber source (or sheared fiber source, etc.) isthen “exploded” when the pressure is released. The condensed moistureinstantaneously evaporates due to the sudden decrease in pressure andthe expansion of the water vapor exerts a shear force upon the fibersource (or sheared fiber source, etc.). A sufficient shear force willcause the mechanical breakdown of the internal structures (e.g., thelignocellulosic structures) of the fiber source.

The sheared and steam exploded fibrous material is then converted into auseful product, such as ethanol. In some embodiments, the fibrousmaterial is converted into a fuel. One method of converting the fibrousmaterial into a fuel is by hydrolysis to produce fermentable sugars,412, which are then fermented to produce the product. Other known andunknown methods of converting fibrous materials into fuels may also beused.

In some embodiments, prior to combining the microorganism, the shearedand steam exploded fibrous material 401 is sterilized to kill anycompeting microorganisms that may be on the fibrous material. Forexample, the fibrous material can be sterilized by exposing the fibrousmaterial to radiation, such as infrared radiation, ultravioletradiation, or an ionizing radiation, such as gamma radiation. Themicroorganisms can also be killed using chemical sterilants, such asbleach (e.g., sodium hypochlorite), chlorhexidine, or ethylene oxide.

One method to hydrolyze the sheared and steam exploded fibrous materialis by the use of cellulases. Cellulases are a group of enzymes that actsynergistically to hydrolyze cellulose. Commercially availableACCELLERASE® 1000 enzyme complex, which contains a complex of enzymesthat reduces lignocellulosic biomass into fermentable sugars can also beused.

According to current understanding, the components of cellulase includeendoglucanases, exoglucanases (cellobiohydrolases), and b-glucosidases(cellobiases). Synergism between the cellulase components exists whenhydrolysis by a combination of two or more components exceeds the sum ofthe activities expressed by the individual components. The generallyaccepted mechanism of a cellulase system (particularly of T.longibrachiatum) on crystalline cellulose is: endoglucanase hydrolyzesinternal B-1,4-glycosidic bonds of the amorphous regions, therebyincreasing the number of exposed non-reducing ends. Exoglucanases thencleave off cellobiose units from the nonreducing ends, which in turn arehydrolyzed to individual glucose units by β-glucosidases. There areseveral configurations of both endo- and exo-glucanases differing instereospecificities. In general, the synergistic action of thecomponents in various configurations is required for optimum cellulosehydrolysis. Cellulases, however, are more inclined to hydrolyze theamorphous regions of cellulose. A linear relationship betweencrystallinity and hydrolysis rates exists whereby higher crystallinityindices correspond to slower enzyme hydrolysis rates. Amorphous regionsof cellulose hydrolyze at twice the rate of crystalline regions. Thehydrolysis of the sheared and steam exploded fibrous material may beperformed by any hydrolyzing biomass process.

Steam explosion of biomass sometimes causes the formation ofby-products, e.g., toxicants, that are inhibitory to microbial andenzymatic activities. The process of converting the sheared and steamexploded fibrous material into a fuel can therefore optionally includean overliming step prior to fermentation to precipitate some of thetoxicants. For example, the pH of the sheared and steam exploded fibrousmaterial may be raised to exceed the pH of 10 by adding calciumhydroxide (Ca(OH)₂) followed by a step of lowering the pH to about 5 byadding H₂SO₄. The overlimed fibrous material may then be used as iswithout the removal of precipitates. As shown in FIG. 23, the optionaloverliming step occurs just prior to the step of hydrolysis of thesheared and steam exploded fibrous material, but it is also contemplatedto perform the overliming step after the hydrolysis step and prior tothe fermenting step.

FIG. 24 depicts an example of a steam explosion apparatus 460. The steamexplosion apparatus 460 includes a reaction chamber 462, in which thefiber source and/or the fibrous material placed through a fiber sourceinlet 464. The reaction chamber is sealed by closing fiber source inletvalve 465. The reaction chamber further includes a pressurized steaminlet 466 that includes a steam valve 467. The reaction chamber furtherincludes an explosive depressurization outlet 468 that includes anoutlet valve 469 in communication with the cyclone 470 through theconnecting pipe 472. Once the reaction chamber includes the fiber sourceand/or sheared fiber source and is sealed by closing valves 465, 467 and469, steam is delivered into the reaction chamber 462 by opening thesteam inlet valve 467 allowing steam to travel through steam inlet 466.Once the reaction chamber reaches target temperature, which can takeabout 20-60 seconds, the holding time begins. The reaction temperatureis held at the target temperature for the desired holding time, whichtypically lasts from about 10 seconds to 5 minutes. At the end of theholding time period, outlet valve is open to allow for explosivedepressurization to occur. The process of explosive depressurizationpropels the contents of the reaction chamber 462 out of the explosivedepressurization outlet 468, through the connecting pipe 472, and intothe cyclone 470. The steam exploded fiber source or fibrous materialthen exits the cyclone in a sludge form into the collection bin 474 asmuch of the remaining steam exits the cyclone into the atmospherethrough vent 476. The steam explosion apparatus further includes washoutlet 478 with wash outlet valve 479 in communication with connectingpipe 472. The wash outlet valve 479 is closed during the use of thesteam explosion apparatus 460 for steam explosion, but opened during thewashing of the reaction chamber 462. The target temperature of thereaction chamber 462 is preferably between 180 and 240 degrees Celsiusor between 200 and 220 degrees Celsius. The holding time is preferablybetween 10 seconds and 30 minutes, or between 30 seconds and 10 minutes,or between 1 minute and 5 minutes.

Because the steam explosion process results in a sludge of steamexploded fibrous material, the steam exploded fibrous material mayoptionally include a fiber recovery process where the “liquor” isseparated from the steam exploded fibrous material. This fiber recoverystep is helpful in that it enables further shearing and/or screeningprocesses and can allow for the conversion of the fibrous material intofuel. The fiber recovery process occurs through the use of a mesh clothto separate the fibers from the liquor. Further drying processes canalso be included to prepare the fibrous material or steam exploded fibersource for subsequent processing.

Any processing technique described herein can be used at pressure aboveor below normal, earth-bound atmospheric pressure. For example, anyprocess that utilizes radiation, sonication, oxidation, pyrolysis, steamexplosion, or combinations of any of these processes to providematerials that include a carbohydrate can be performed under highpressure, which, can increase reaction rates. For example, any processor combination of processes can be performed at a pressure greater thanabout greater than 25 MPa, e.g., greater than 50 MPa, 75 MPa, 100 MPa,150 MPa, 200 MPa, 250 MPa, 350 MPa, 500 MPa, 750 MPa, 1,000 MPa, orgreater than 1,500 MPa.

Combinations of Irradiating, Sonicating, and Oxidizing Devices

In some embodiments, it may be advantageous to combine two or moreseparate irradiation, sonication, pyrolization, and/or oxidation devicesinto a single hybrid machine. For such a hybrid machine, multipleprocesses may be performed in close juxtaposition or evensimultaneously, with the benefit of increasing pretreatment throughputand potential cost savings.

For example, consider the electron beam irradiation and sonicationprocesses. Each separate process is effective in lowering the meanmolecular weight of cellulosic material by an order of magnitude ormore, and by several orders of magnitude when performed serially.

Both irradiation and sonication processes can be applied using a hybridelectron beam/sonication device as is illustrated in FIG. 25. Hybridelectron beam/sonication device 2500 is pictured above a shallow pool(depth ˜3-5 cm) of a slurry of cellulosic material 2550 dispersed in anaqueous, oxidant medium, such as hydrogen peroxide or carbamideperoxide. Hybrid device 2500 has an energy source 2510, which powersboth electron beam emitter 2540 and sonication horns 2530.

Electron beam emitter 2540 generates electron beams which pass though anelectron beam aiming device 2545 to impact the slurry 2550 containingcellulosic material. The electron beam aiming device can be a scannerthat sweeps a beam over a range of up to about 6 feet in a directionapproximately parallel to the surface of the slurry 2550.

On either side of the electron beam emitter 2540 are sonication horns2530, which deliver ultrasonic wave energy to the slurry 2550. Thesonication horns 2530 end in a detachable endpiece 2535 that is incontact with the slurry 2550.

The sonication horns 2530 are at risk of damage from long-term residualexposure to the electron beam radiation. Thus, the horns can beprotected with a standard shield 2520, e.g., made of lead or aheavy-metal-containing alloy such as Lipowitz metal, which is imperviousto electron beam radiation. Precautions must be taken, however, toensure that the ultrasonic energy is not affected by the presence of theshield. The detachable endpieces 2535, are constructed of the samematerial and attached to the horns 2530, are used to be in contact withthe cellulosic material 2550 and are expected to be damaged.Accordingly, the detachable endpieces 2535 are constructed to be easilyreplaceable.

A further benefit of such a simultaneous electron beam and ultrasoundprocess is that the two processes have complementary results. Withelectron beam irradiation alone, an insufficient dose may result incross-linking of some of the polymers in the cellulosic material, whichlowers the efficiency of the overall depolymerization process. Lowerdoses of electron beam irradiation and/or ultrasound radiation may alsobe used to achieve a similar degree of depolymerization as that achievedusing electron beam irradiation and sonication separately.

An electron beam device can also be combined with one or more ofhigh-frequency, rotor-stator devices, which can be used as analternative to ultrasonic energy devices, and performs a similarfunction.

Further combinations of devices are also possible. For example, anionizing radiation device that produces gamma radiation emitted from,e.g., ⁶⁰Co pellets, can be combined with an electron beam source and/oran ultrasonic wave source. Shielding requirements may be more stringentin this case.

The radiation devices for pretreating biomass discussed above can alsobe combined with one or more devices that perform one or more pyrolysisprocessing sequences. Such a combination may again have the advantage ofhigher throughput.

Nevertheless, caution must be observed, as there may be conflictingrequirements between some radiation processes and pyrolysis. Forexample, ultrasonic radiation devices may require the feedstock beimmersed in a liquid oxidizing medium. On the other hand, as discussedpreviously, it may be advantageous for a sample of feedstock undergoingpyrolysis to be of a particular moisture content. In this case, the newsystems automatically measure and monitor for a particular moisturecontent and regulate the same Further, some or all of the above devices,especially the pyrolysis device, can be combined with an oxidationdevice as discussed previously.

Primary Processes Fermentation

Generally, various microorganisms can produce a number of usefulproducts, such as a fuel, by operating on, e.g., fermenting thepretreated biomass materials. For example, Natural Force™ Chemistrymethods can be used to prepare biomass materials for use infermentation. Alcohols, organic acids, hydrocarbons, hydrogen, proteinsor mixtures of any of these materials, for example, can be produced byfermentation or other processes.

The microorganism can be a natural microorganism or an engineeredmicroorganism. For example, the microorganism can be a bacterium, e.g.,a cellulolytic bacterium, a fungus, e.g., a yeast, a plant or a protist,e.g., an algae, a protozoa or a fungus-like protist, e.g., a slime mold.When the organisms are compatible, mixtures of organisms can beutilized.

To aid in the breakdown of the materials that include the cellulose, oneor more enzymes, e.g., a cellulolytic enzyme can be utilized. In someembodiments, the materials that include the cellulose are first treatedwith the enzyme, e.g., by combining the material and the enzyme in anaqueous solution. This material can then be combined with themicroorganism. In other embodiments, the materials that include thecellulose, the one or more enzymes and the microorganism are combined atthe concurrently, e.g., by combining in an aqueous solution.

Also, to aid in the breakdown of the materials that include thecellulose, the materials can be treated post irradiation with heat, achemical (e.g., mineral acid, base or a strong oxidizer such as sodiumhypochlorite), and/or an enzyme.

During the fermentation, sugars released from cellulolytic hydrolysis orthe saccharification step, are fermented to, e.g., ethanol, by afermenting microorganism such as yeast. Suitable fermentingmicroorganisms have the ability to convert carbohydrates, such asglucose, xylose, arabinose, mannose, galactose, oligosaccharides orpolysaccharides into fermentation products. Fermenting microorganismsinclude strains of the genus Saccharomyces spp. e.g., Saccharomycescerevisiae (baker's yeast), Saccharomyces distaticus, Saccharomycesuvarum; the genus Kluyveromyces, e.g., species Kluyveromyces marxianus,Kluyveromyces fragilis; the genus Candida, e.g., Candidapseudotropicalis, and Candida brassicae, Pichia stipitis (a relative ofCandida shehatae, the genus Clavispora, e.g., species Clavisporalusitaniae and Clavispora opuntiae the genus Pachysolen, e.g., speciesPachysolen tannophilus, the genus Bretannomyces, e.g., speciesBretannomyces clausenii (Philippidis, G. P., 1996, Cellulosebioconversion technology, in Handbook on Bioethanol: Production andUtilization, Wyman, C. E., ed., Taylor & Francis, Washington, D.C.,179-212).

Commercially available yeast include, for example, Red Star®/LesaffreEthanol Red (available from Red Star/Lesaffre, USA) FALI® (availablefrom Fleischmann's Yeast, a division of Burns Philip Food Inc., USA),SUPERSTART® (available from Alltech, now Lallemand), GERT STRAND®(available from Gert Strand AB, Sweden) and FERMOL® (available from DSMSpecialties).

Bacteria that can ferment bimoss to ethanol and other products include,e.g., Zymomonas mobilis and Clostridium thermocellum (Philippidis, 1996,supra). Leschine et al. (International Journal of Systematic andEvolutionary Microbiology 2002, 52, 1155-1160) isolated an anaerobic,mesophilic, cellulolytic bacterium from forest soil, Clostridiumphytofermentans sp. nov., which converts cellulose to ethanol.

Fermentation of biomass to ethanol and other products may be carried outusing certain types of thermophilic or genetically engineeredmicroorganisms, such Thermoanaerobacter species, including T. mathranii,and yeast species such as Pichia species. An example of a strain of T.mathranii is A3M4 described in Sonne-Hansen et al. (Applied Microbiologyand Biotechnology 1993, 38, 537-541) or Ahring et al. (Arch. Microbiol.1997, 168, 114-119).

Yeast and Zymomonas bacteria can be used for fermentation or conversion.The optimum pH for yeast is from about pH 4 to 5, while the optimum pHfor Zymomonas is from about pH 5 to 6. Typical fermentation times areabout 24 to 96 hours with temperatures in the range of 26° C. to 40° C.,however thermophilic microorganisms prefer higher temperatures.

Enzymes which break down biomass, such as cellulose, to lower molecularweight carbohydrate-containing materials, such as glucose, duringsaccharification are referred to as cellulolytic enzymes or cellulase.These enzymes may be a complex of enzymes that act synergistically todegrade crystalline cellulose. Examples of cellulolytic enzymes include:endoglucanases, cellobiohydrolases, and cellobiases (β-glucosidases). Acellulosic substrate is initially hydrolyzed by endoglucanases at randomlocations producing oligomeric intermediates. These intermediates arethen substrates for exo-splitting glucanases such as cellobiohydrolaseto produce cellobiose from the ends of the cellulose polymer. Cellobioseis a water-soluble B-1,4-linked dimer of glucose. Finally cellobiasecleaves cellobiose to yield glucose.

A cellulase is capable of degrading biomass and may be of fungal orbacterial origin. Suitable enzymes include cellulases from the generaBacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium,Chrysosporium and Trichoderma, and include species of Humicola,Coprinus, Thielavia, Fusarium, Myceliophthora, Acremonium,Cephalosporium, Scytalidium, Penicillium or Aspergillus (see, e.g., EP458162), especially those produced by a strain selected from the speciesHumicola insolens (reclassified as Scytalidium thermophilum, see, e.g.,U.S. Pat. No. 4,435,307), Coprinus cinereus, Fusarium oxysporum,Myceliophthora thermophile, Meripilus giganteus, Thielavia terrestris,Acremonium sp., Acremonium persicinum, Acremonium acremonium, Acremoniumbrachypenium, Acremonium dichromosporum, Acremonium obclavatum,Acremonium pinkertoniae, Acremonium roseogriseum, Acremoniumincoloratum, and Acremonium furatum; preferably from the speciesHumicola insolens DSM 1800, Fusarium oxysporum DSM 2672, Myceliophthorathermophile CBS 117.65, Cephalosporium sp. RYM-202, Acremonium sp. CBS478.94, Acremonium sp. CBS 265.95, Acremonium persicinum CBS 169.65,Acremonium acremonium AHU 9519, Cephalosporium sp. CBS 535.71,Acremonium brachypenium CBS 866.73, Acremonium dichromosporum CBS683.73, Acremonium obclavatum CBS 311.74, Acremonium pinkertoniae CBS157.70, Acremonium roseogriseum CBS 134.56, Acremonium incoloratum CBS146.62, and Acremonium furatum CBS 299.70H. Cellulolytic enzymes mayalso be obtained from Chrysosporium, preferably a strain ofChrysosporium lucknowense. Additionally, Trichoderma (particularlyTrichoderma viride, Trichoderma reesei, and Trichoderma koningii),alkalophilic Bacillus (see, for example, U.S. Pat. No. 3,844,890 and EP458162), and Streptomyces (see, e.g., EP 458162) may be used. Thebacterium, Saccharophagus degradans, produces a mixture of enzymescapable of degrading a range of cellulosic materials and may also beused in this process.

Anaerobic cellulolytic bacteria have also been isolated from soil, e.g.,a novel cellulolytic species of Clostiridium, Clostridiumphytofermentans sp. nov. (see Leschine et. al, International Journal ofSystematic and Evolutionary Microbiology (2002), 52, 1155-1160).

Cellulolytic enzymes using recombinant technology can also be used (see,e.g., WO 2007/071818 and WO 2006/110891).

The cellulolytic enzymes used can be produced by fermentation of theabove-noted microbial strains on a nutrient medium containing suitablecarbon and nitrogen sources and inorganic salts, using procedures knownin the art (see, e.g., Bennett, J. W. and LaSure, L. (eds.), More GeneManipulations in Fungi, Academic Press, C A 1991). Suitable media areavailable from commercial suppliers or may be prepared according topublished compositions (e.g., in catalogues of the American Type CultureCollection). Temperature ranges and other conditions suitable for growthand cellulase production are known in the art (see, e.g., Bailey, J. E.,and Ollis, D. F., Biochemical Engineering Fundamentals, McGraw-Hill BookCompany, N Y, 1986).

Treatment of cellulose with cellulase is usually carried out attemperatures between 30° C. and 65° C. Cellulases are active over arange of pH of about 3 to 7. A saccharification step may last up to 120hours. The cellulase enzyme dosage achieves a sufficiently high level ofcellulose conversion. For example, an appropriate cellulase dosage istypically between 5.0 and 50 Filter Paper Units (FPU or IU) per gram ofcellulose. The FPU is a standard measurement and is defined and measuredaccording to Ghose (1987, Pure and Appl. Chem. 59:257-268).

Mobile fermentors can be utilized, as described in U.S. ProvisionalPatent Application Ser. 60/832,735, now Published InternationalApplication No. WO 2008/011598.

Gasification

In addition to using pyrolysis for pre-treatment of feedstock, pyrolysiscan also be used to process pre-treated feedstock to extract usefulmaterials. In particular, a form of pyrolysis known as gasification canbe employed to generate fuel gases along with various other gaseous,liquid, and solid products. To perform gasification, the pre-treatedfeedstock is introduced into a pyrolysis chamber and heated to a hightemperature, typically 700° C. or more. The temperature used dependsupon a number of factors, including the nature of the feedstock and thedesired products.

Quantities of oxygen (e.g., as pure oxygen gas and/or as air) and steam(e.g., superheated steam) are also added to the pyrolysis chamber tofacilitate gasification. These compounds react with carbon-containingfeedstock material in a multiple-step reaction to generate a gas mixturecalled synthesis gas (or “syngas”). Essentially, during gasification, alimited amount of oxygen is introduced into the pyrolysis chamber toallow some feedstock material to combust to form carbon monoxide andgenerate process heat. The process heat can then be used to promote asecond reaction that converts additional feedstock material to hydrogenand carbon monoxide.

In a first step of the overall reaction, heating the feedstock materialproduces a char that can include a wide variety of differenthydrocarbon-based species. Certain volatile materials can be produced(e.g., certain gaseous hydrocarbon materials), resulting in a reductionof the overall weight of the feedstock material. Then, in a second stepof the reaction, some of the volatile material that is produced in thefirst step reacts with oxygen in a combustion reaction to produce bothcarbon monoxide and carbon dioxide. The combustion reaction releasesheat, which promotes the third step of the reaction. In the third step,carbon dioxide and steam (e.g., water) react with the char generated inthe first step to form carbon monoxide and hydrogen gas. Carbon monoxidecan also react with steam, in a water gas shift reaction, to form carbondioxide and further hydrogen gas.

Gasification can be used as a primary process to generate productsdirectly from pre-treated feedstock for subsequent transport and/orsale, for example. Alternatively, or in addition, gasification can beused as an auxiliary process for generating fuel for an overallprocessing system. The hydrogen-rich syngas that is generated via thegasification process can be burned, for example, to generate electricityand/or process heat that can be directed for use at other locations inthe processing system. As a result, the overall processing system can beat least partially self-sustaining. A number of other products,including pyrolysis oils and gaseous hydrocarbon-based substances, canalso be obtained during and/or following gasification; these can beseparated and stored or transported as desired.

A variety of different pyrolysis chambers are suitable for gasificationof pre-treated feedstock, including the pyrolysis chambers disclosedherein. In particular, fluidized bed reactor systems, in which thepre-treated feedstock is fluidized in steam and oxygen/air, providerelatively high throughput and straightforward recovery of products.Solid char that remains following gasification in a fluidized bed system(or in other pyrolysis chambers) can be burned to generate additionalprocess heat to promote subsequent gasification reactions.

Post-Processing Distillation

After fermentation, the resulting fluids can be distilled using, forexample, a “beer column” to separate ethanol and other alcohols from themajority of water and residual solids. The vapor exiting the beer columncan be 35% by weight ethanol and fed to a rectification column. Amixture of nearly azeotropic (92.5%) ethanol and water from therectification column can be purified to pure (99.5%) ethanol usingvapor-phase molecular sieves. The beer column bottoms can be sent to thefirst effect of a three-effect evaporator. The rectification columnreflux condenser can provide heat for this first effect. After the firsteffect, solids can be separated using a centrifuge and dried in a rotarydryer. A portion (25%) of the centrifuge effluent can be recycled tofermentation and the rest sent to the second and third evaporatoreffects. Most of the evaporator condensate can be returned to theprocess as fairly clean condensate with a small portion split off towaste water treatment to prevent build-up of low-boiling compounds.

Waste Water Treatment

Wastewater treatment is used to minimize makeup water requirements ofthe plant by treating process water for reuse within the plant.Wastewater treatment can also produce fuel (e.g., sludge and biogas)that can be used to improve the overall efficiency of the ethanolproduction process. For example, as described in further detail below,sludge and biogas can be used to create steam and electricity that canbe used in various plant processes.

Wastewater is initially pumped through a screen (e.g., a bar screen) toremove large particles, which are collected in a hopper. In someembodiments, the large particles are sent to a landfill. Additionally oralternatively, the large particles are burned to create steam and/orelectricity as described in further detail below. In general, thespacing on the bar screen is between ¼ inch to 1 inch spacing (e.g., ½inch spacing).

The wastewater then flows to an equalization tank, where the organicconcentration of the wastewater is equalized during a retention time. Ingeneral, the retention time is between 8 hours and 36 hours (e.g., 24hours). A mixer is disposed within the tank to stir the contents of thetank. In some embodiments, a plurality of mixers disposed throughout thetank are used to stir the contents of the tank. In certain embodiments,the mixer substantially mixes the contents of the equalization tank suchthat conditions (e.g., wastewater concentration and temperature)throughout the tank are uniform.

A first pump moves water from the equalization tank through aliquid-to-liquid heat exchanger. The heat exchanger is controlled (e.g.,by controlling the flow rate of fluid through the heat exchanger) suchthat wastewater exiting the heat exchanger is at a desired temperaturefor anaerobic treatment. For example, the desired temperature foranaerobic treatment can be between 40° C. to 60° C.

After exiting the heat exchanger, the wastewater enters one or moreanaerobic reactors. In some embodiments, the concentration of sludge ineach anaerobic reactor is the same as the overall concentration ofsludge in the wastewater. In other embodiments, the anaerobic reactorhas a higher concentration of sludge than the overall concentration ofsludge in the wastewater.

A nutrient solution containing nitrogen and phosphorus is metered intoeach anaerobic reactor containing wastewater. The nutrient solutionreacts with the sludge in the anaerobic reactor to produce biogas whichcan contain 50% methane and have a heating value of approximately 12,000British thermal units, or Btu, per pound). The biogas exits eachanaerobic reactor through a vent and flows into a manifold, where aplurality of biogas streams are combined into a single stream. Acompressor moves the stream of biogas to a boiler or a combustion engineas described in further detail below. In some embodiments, thecompressor also moves the single stream of biogas through adesulphurization catalyst. Additionally or alternatively, the compressorcan move the single stream of biogas through a sediment trap.

A second pump moves anaerobic effluent from the anaerobic reactors toone or more aerobic reactors (e.g., activated sludge reactors). Anaerator is disposed within each aerobic reactor to mix the anaerobiceffluent, sludge, and oxygen (e.g., oxygen contained in air). Withineach aerobic reactor, oxidation of cellular material in the anaerobiceffluent produces carbon dioxide, water, and ammonia.

Aerobic effluent moves (e.g., via gravity) to a separator, where sludgeis separated from treated water. Some of the sludge is returned to theone or more aerobic reactors to create an elevated sludge concentrationin the aerobic reactors, thereby facilitating the aerobic breakdown ofcellular material in the wastewater. A conveyor removes excess sludgefrom the separator. As described in further detail below, the excesssludge is used as fuel to create steam and/or electricity.

The treated water is pumped from the separator to a settling tank.Solids dispersed throughout the treated water settle to the bottom ofthe settling tank and are subsequently removed. After a settling period,treated water is pumped from the settling tank through a fine filter toremove any additional solids remaining in the water. In someembodiments, chlorine is added to the treated water to kill pathogenicbacteria. In some embodiments, one or more physical-chemical separationtechniques are used to further purify the treated water. For example,treated water can be pumped through a carbon adsorption reactor. Asanother example, treated water can pumped through a reverse osmosisreactor.

Waste Combustion

The production of alcohol from biomass can result in the production ofvarious by-product streams useful for generating steam and electricityto be used in other parts of the plant. For example, steam generatedfrom burning by-product streams can be used in the distillation process.As another example, electricity generated from burning by-productstreams can be used to power electron beam generators and ultrasonictransducers used in pretreatment.

The by-products used to generate steam and electricity are derived froma number of sources throughout the process. For example, anaerobicdigestion of wastewater produces a biogas high in methane and a smallamount of waste biomass (sludge). As another example, post-distillatesolids (e.g., unconverted lignin, cellulose, and hemicellulose remainingfrom the pretreatment and primary processes) can be used as a fuel.

The biogas is diverted to a combustion engine connected to an electricgenerator to produce electricity. For example, the biogas can be used asa fuel source for a spark-ignited natural gas engine. As anotherexample, the biogas can be used as a fuel source for a direct-injectionnatural gas engine. As another example, the biogas can be used as a fuelsource for a combustion turbine. Additionally or alternatively, thecombustion engine can be configured in a cogeneration configuration. Forexample, waste heat from the combustion engines can be used to providehot water or steam throughout the plant.

The sludge, and post-distillate solids are burned to heat water flowingthrough a heat exchanger. In some embodiments, the water flowing throughthe heat exchanger is evaporated and superheated to steam. In certainembodiments, the steam is used in the pretreatment rector and in heatexchange in the distillation and evaporation processes. Additionally oralternatively, the steam expands to power a multi-stage steam turbineconnected to an electric generator. Steam exiting the steam turbine iscondensed with cooling water and returned to the heat exchanger forreheating to steam. In some embodiments, the flow rate of water throughthe heat exchanger is controlled to obtain a target electricity outputfrom the steam turbine connected to an electric generator. For example,water can be added to the heat exchanger to ensure that the steamturbine is operating above a threshold condition (e.g., the turbine isspinning fast enough to turn the electric generator).

While certain embodiments have been described, other embodiments arepossible.

As an example, while the biogas is described as being diverted to acombustion engine connected to an electric generator, in certainembodiments, the biogas can be passed through a fuel reformer to producehydrogen. The hydrogen is then converted to electricity through a fuelcell.

As another example, while the biogas is described as being burned apartfrom the sludge and post-distillate solids, in certain embodiments, allof the waste by-products can be burned together to produce steam.

Products/Co-Products Alcohols

The alcohol produced can be a monohydroxy alcohol, e.g., ethanol, or apolyhydroxy alcohol, e.g., ethylene glycol or glycerin. Examples ofalcohols that can be produced include methanol, ethanol, propanol,isopropanol, butanol, e.g., n-, sec- or t-butanol, ethylene glycol,propylene glycol, 1,4-butane diol, glycerin or mixtures of thesealcohols.

Each of the alcohols produced by the plant have commercial value asindustrial feedstock. For example, ethanol can be used in themanufacture of varnishes and perfume. As another example, methanol canbe used as a solvent used as a component in windshield wiper fluid. Asstill another example, butanol can be used in plasticizers, resins,lacquers, and brake fluids.

Bioethanol produced by the plant is valuable as an ingredient used inthe food and beverage industry. For example, the ethanol produced by theplant can be purified to food grade alcohol and used as a primaryingredient in the alcoholic beverages.

Bioethanol produced by the plant also has commercial value as atransportation fuel. The use of ethanol as a transportation fuel can beimplemented with relatively little capital investment from sparkignition engine manufacturers and owners (e.g., changes to injectiontiming, fuel-to-air ratio, and components of the fuel injection system).Many automotive manufacturers currently offer flex-fuel vehicles capableof operation on ethanol/gasoline blends up to 85% ethanol by volume(e.g., standard equipment on a Chevy Tahoe 4×4).

Fuels and other products (e.g., ethanol, bioethanol, other alcohols, andother combustible hydrocarbons) produced via the methods disclosedherein can be blended with other hydrocarbon-containing species. Forexample, ethanol produced using any of the methods disclosed herein canbe blended with gasoline to produce “gasohol,” which can be used ascombustible fuel in a wide variety of applications, including automobileengines.

Bioethanol produced by this plant can be used as an engine fuel toimprove environmental and economic conditions beyond the location of theplant. For example, ethanol produced by this plant and used as a fuelcan reduce greenhouse gas emissions from manmade sources (e.g.,transportation sources). As another example, ethanol produced by thisplant and used as an engine fuel can also displace consumption ofgasoline refined from oil.

Bioethanol has a greater octane number than conventional gasoline and,thus, can be used to improve the performance (e.g., allow for highercompression ratios) of spark ignition engines. For example, smallamounts (e.g., 10% by volume) of ethanol can be blended with gasoline toact as an octane enhancer for fuels used in spark ignition engines. Asanother example, larger amounts (e.g., 85% by volume) of ethanol can beblended with gasoline to further increase the fuel octane number anddisplace larger volumes of gasoline.

Bioethanol strategies are discussed, e.g., by DiPardo in Journal ofOutlook for Biomass Ethanol Production and Demand (EIA Forecasts), 2002;Sheehan in Biotechnology Progress, 15:8179, 1999; Martin in EnzymeMicrobes Technology, 31:274, 2002; Greer in BioCycle, 61-65, April 2005;Lynd in Microbiology and Molecular Biology Reviews, 66:3, 506-577, 2002;Ljungdahl et al. in U.S. Pat. No. 4,292,406; and Bellamy in U.S. Pat.No. 4,094,742.

Organic Acids

The organic acids produced can include monocarboxylic acids or apolycarboxylic acids. Examples of organic acids include formic acid,acetic acid, propionic acid, butyric acid, valeric acid, caproic,palmitic acid, stearic acid, oxalic acid, malonic acid, succinic acid,glutaric acid, oleic acid, linoleic acid, glycolic acid, lactic acid,γ-hydroxybutyric acid or mixtures of these acids.

Food Products

In some embodiments, all or a portion of the fermentation process can beinterrupted before the cellulosic material is completely converted toethanol. The intermediate fermentation products include highconcentrations of sugar and carbohydrates. These intermediatefermentation products can be used in preparation of food for human oranimal consumption or for use in agriculture or aquaculture. In someembodiments, irradiation pretreatment of the cellulosic material willrender the intermediate fermentation products sterile (e.g., fit forhuman consumption or for use in agriculture or aquaculture). In someembodiments, the intermediate fermentation products will requirepost-processing prior to use as food. For example, a dryer can be usedto remove moisture from the intermediate fermentation products tofacilitate storage, handling, and shelf-life. Additionally oralternatively, the intermediate fermentation products can be ground to afine particle size in a stainless-steel laboratory mill to produce aflour-like substance.

Animal Feed

Distillers grains and solubles can be converted into a valuablebyproduct of the distillation-dehydration process. After thedistillation-dehydration process, distillers grains and solubles can bedried to improve the ability to store and handle the material. Theresulting dried distillers grains and solubles (DDGS) is low in starch,high in fat, high in protein, high in fiber, and high in phosphorous.Thus, for example, DDGS can be valuable as a source of animal feed(e.g., as a feed source for dairy cattle). DDGS can be subsequentlycombined with nutritional additives to meet specific dietaryrequirements of specific categories of animals (e.g., balancingdigestible lysine and phosphorus for swine diets).

Pharmaceuticals

The pretreatment processes discussed above can be applied to plants withmedicinal properties. In some embodiments, sonication can stimulatebioactivity and/or bioavailabilty of the medicinal components of plantswith medicinal properties. Additionally or alternatively, irradiationstimulates bioactivity and/or bioavailabilty of the medicinal componentsof plants with medicinal properties. For example, sonication andirradiation can be combined in the pretreatment of willow bark tostimulate the production of salicin.

Nutriceuticals

In some embodiments, intermediate fermentation products (e.g., productsthat include high concentrations of sugar and carbohydrates) can besupplemented to create a nutriceutical. For example, intermediatefermentation products can be supplemented with calcium to create anutriceutical that provides energy and helps improve or maintain bonestrength.

Co-Products Lignin Residue

As described above, lignin-containing residues from primary andpretreatment processes has value as a high/medium energy fuel and can beused to generate power and steam for use in plant processes. However,such lignin residues are a new type of solids fuel and there is littledemand for it outside of the plant boundaries, and the costs of dryingit for transportation only subtract from its potential value. In somecases, gasification of the lignin residues can convert the residues to ahigher-value product with lower cost.

Other Co-Products

Cell matter, furfural, and acetic acid have been identified as potentialco-products of biomass-to-fuel processing facilities. Interstitial cellmatter could be valuable, but might require significant purification.Markets for furfural and acetic acid are in place, although it isunlikely that they are large enough to consume the output of a fullycommercialized lignocellulose-to-ethanol industry.

Conversion of Starchy Materials

FIGS. 26 and 27 show block diagrams for a dry and wet milling process,respectively, and illustrate the conversion, e.g., fermentation, of cornkernels to ethanol and other valuable co-products.

Referring particularly now to FIG. 26, in some implementations, a drymilling process for the conversion of corn kernels to ethanol, e.g.,anhydrous ethanol, that can be utilized as a fuel, e.g., automobile oraviation fuel, can begin with pretreating the dried corn kernels withany one or more pretreatments described herein, such as radiation, e.g.,any one or more types of radiation described herein (e.g., a beam ofelectrons in which each electron has an energy of about 5 MeV or a beamof protons in which the energy of each proton is about 3-100 MeV). Afterpre-treatment, the corn kernels can be ground and/or sheared into apowder. Although any one or more pretreatments described herein can beapplied after grinding and/or at any time during the dry milling processoutlined in FIG. 26, pretreating prior to grinding and/or shearing canbe advantageous in that the kernels are generally more brittle afterpretreatment and, as a result, are easier and can require less energy togrind and/or shear. In some embodiments, a selected pretreatment can beapplied more than once during conversion, e.g., prior to milling andthen after milling.

After grinding and/or shearing, the milled, dry kernels can beoptionally hydrated by adding the milled material to a vessel containingwater and, optionally, hydrating agents, such as surfactants.Optionally, this reaction vessel can also include one or more enzymes,such as amylase, to aid in further breakdown of starchy biomass, or thereaction vessel may contain one or more acids, such as a mineral acid,e.g., dilute sulfuric acid. If a hydration vessel is utilized, itscontents are emptied into a conversion vessel, e.g., a fermentationvessel, that includes one or more conversion microbes, such as one ormore yeasts, bacteria or mixtures of yeasts and/or bacteria. If ahydration vessel is not utilized, the milled material can be directlycharged to the conversion vessel, e.g., for fermentation.

After conversion, the remaining solids are removed and dried to givedistillers dry grains (DDG), while the ethanol is distilled off. In someembodiments, a thermophilic microbe is utilized for the conversion andthe ethanol is continuously removed by evaporation as it is produced. Ifdesired, the distilled ethanol can be fully dehydrated, such as bypassing the wet ethanol through a zeolite bed, or distilling withbenzene.

Referring particularly now to FIG. 27, in some implementations, the wetmilling process for the conversion of corn kernels to anhydrous ethanolbegins with pretreating the dried corn kernels with any one or morepretreatments described herein, such as radiation, e.g., any one or moretypes of radiation described herein (e.g., a beam of electrons in whicheach electron has an energy of about 5 MeV). After pre-treatment, thecorn kernels are steeped in dilute sulfuric acid and gently stirred tobreak the corn kernels into its constituents. After steeping, the fiber,oil and germ portions are fractionated and dried, and then combined withany solids remaining after distillation to give corn gluten feed (CGF).After removing germ and fiber, in some embodiments, the gluten isseparated to give corn gluten meal (CGM). The remaining starch can bepretreated again (or for the first time) by any pretreatment describedherein, e.g., to reduce its molecular weight and/or to functionalize thestarch so that it is more soluble. In some embodiments, the starch isthen charged to a reaction vessel containing water and, optionally,hydrating agents, such as surfactants. Optionally, this reaction vesselcan also include one or more enzymes, such as amylase, to aid in furtherbreakdown of starch, or the reaction vessel may contain one or moreacids, such as a mineral acid, e.g., dilute sulfuric acid. As shown,saccharification can occur in several vessels and then the contents ofthe final vessel can be emptied into a conversion vessel, e.g., afermentation vessel, that includes one or more conversion microbes, suchas one or more yeasts or bacteria.

After conversion, the ethanol is distilled off. In some embodiments, athermophilic microbe is utilized for the conversion and the ethanol iscontinuously removed by evaporation as it is produced. If desired, thedistilled ethanol can be fully dehydrated, such as by passing the wetethanol through a zeolite bed.

EXAMPLES

The following Examples are intended to illustrate, and do not limit theteachings of this disclosure.

Example 1 Preparation of Fibrous Material from Polycoated Paper

A 1500 pound skid of virgin, half-gallon juice cartons made ofun-printed polycoated white Kraft board having a bulk density of 20lb/ft³ was obtained from International Paper. Each carton was foldedflat, and then fed into a 3 hp Flinch Baugh shredder at a rate ofapproximately 15 to 20 pounds per hour. The shredder was equipped withtwo 12 inch rotary blades, two fixed blades and a 0.30 inch dischargescreen. The gap between the rotary and fixed blades was adjusted to 0.10inch. The output from the shredder resembled confetti having a width ofbetween 0.1 inch and 0.5 inch, a length of between 0.25 inch and 1 inchand a thickness equivalent to that of the starting material (about 0.075inch).

The confetti-like material was fed to a Munson rotary knife cutter,Model SC30. Model SC30 is equipped with four rotary blades, four fixedblades, and a discharge screen having ⅛ inch openings. The gap betweenthe rotary and fixed blades was set to approximately 0.020 inch. Therotary knife cutter sheared the confetti-like pieces across theknife-edges, tearing the pieces apart and releasing a fibrous materialat a rate of about one pound per hour. The fibrous material had a BETsurface area of 0.9748 m²/g+/−0.0167 m²/g, a porosity of 89.0437 percentand a bulk density (@0.53 psia) of 0.1260 g/mL. An average length of thefibers was 1.141 mm and an average width of the fibers was 0.027 mm,giving an average L/D of 42:1. A scanning electron micrograph of thefibrous material is shown in FIG. 26 at 25× magnification.

Example 2 Preparation of Fibrous Material from Bleached Kraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confettihaving a width of between 0.1 inch and 0.5 inch, a length of between0.25 inch and 1 inch and a thickness equivalent to that of the startingmaterial (about 0.075 inch). The confetti-like material was fed to aMunson rotary knife cutter, Model SC30. The discharge screen had ⅛ inchopenings. The gap between the rotary and fixed blades was set toapproximately 0.020 inch. The rotary knife cutter sheared theconfetti-like pieces, releasing a fibrous material at a rate of aboutone pound per hour. The fibrous material had a BET surface area of1.1316 m²/g+/−0.0103 m²/g, a porosity of 88.3285 percent and a bulkdensity (@0.53 psia) of 0.1497 g/mL. An average length of the fibers was1.063 mm and an average width of the fibers was 0.0245 mm, giving anaverage L/D of 43:1. A scanning electron micrographs of the fibrousmaterial is shown in FIG. 29 at 25× magnification.

Example 3 Preparation of Twice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had 1/16 inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter the confetti-like pieces, releasing a fibrousmaterial at a rate of about one pound per hour. The material resultingfrom the first shearing was fed back into the same setup described aboveand sheared again. The resulting fibrous material had a BET surface areaof 1.4408 m²/g+/−0.0156 m²/g, a porosity of 90.8998 percent and a bulkdensity (@0.53 psia) of 0.1298 g/mL. An average length of the fibers was0.891 mm and an average width of the fibers was 0.026 mm, giving anaverage L/D of 34:1. A scanning electron micrograph of the fibrousmaterial is shown in FIG. 30 at 25× magnification.

Example 4 Preparation of Thrice Sheared Fibrous Material from BleachedKraft Board

A 1500 pound skid of virgin bleached white Kraft board having a bulkdensity of 30 lb/ft³ was obtained from International Paper. The materialwas folded flat, and then fed into a 3 hp Flinch Baugh shredder at arate of approximately 15 to 20 pounds per hour. The shredder wasequipped with two 12 inch rotary blades, two fixed blades and a 0.30inch discharge screen. The gap between the rotary and fixed blades wasadjusted to 0.10 inch. The output from the shredder resembled confetti(as above). The confetti-like material was fed to a Munson rotary knifecutter, Model SC30. The discharge screen had ⅛ inch openings. The gapbetween the rotary and fixed blades was set to approximately 0.020 inch.The rotary knife cutter sheared the confetti-like pieces across theknife-edges. The material resulting from the first shearing was fed backinto the same setup and the screen was replaced with a 1/16 inch screen.This material was sheared. The material resulting from the secondshearing was fed back into the same setup and the screen was replacedwith a 1/32 inch screen. This material was sheared. The resultingfibrous material had a BET surface area of 1.6897 m²/g+/−0.0155 m²/g, aporosity of 87.7163 percent and a bulk density (@0.53 psia) of 0.1448g/mL. An average length of the fibers was 0.824 mm and an average widthof the fibers was 0.0262 mm, giving an average L/D of 32:1. A scanningelectron micrograph of the fibrous material is shown in FIG. 31 at 25×magnification.

Example 5 Preparation of Densified Fibrous Material from Bleached KraftBoard without Added Binder

Fibrous material was prepared according to Example 2. Approximately 1 lbof water was sprayed onto each 10 lb of fibrous material. The fibrousmaterial was densified using a California Pellet Mill 1100 operating at75° C. Pellets were obtained having a bulk density ranging from about 7lb/ft³ to about 15 lb/ft³.

Example 6 Preparation of Densified Fibrous Material from Bleached KraftBoard with Binder

Fibrous material was prepared according to Example 2.

A 2 weight percent stock solution of POLYOX™ WSR N10 (polyethyleneoxide) was prepared in water.

Approximately 1 lb of the stock solution was sprayed onto each 10 lb offibrous material. The fibrous material was densified using a CaliforniaPellet Mill 1100 operating at 75° C. Pellets were obtained having a bulkdensity ranging from about 15 lb/ft³ to about 40 lb/ft³.

Example 7 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Minimum Oxidation

Fibrous material is prepared according to Example 4. The fibrous Kraftpaper is densified according to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is evacuated under high vacuum(10⁻⁵ ton) for 30 minutes, and then back-filled with argon gas. Theampoule is sealed under argon. The pellets in the ampoule are irradiatedwith gamma radiation for about 3 hours at a dose rate of about 1 Mradper hour to provide an irradiated material in which the cellulose has alower molecular weight than the fibrous Kraft starting material.

Example 8 Reducing the Molecular Weight of Cellulose in Fibrous KraftPaper by Gamma Radiation with Maximum Oxidation

Fibrous material is prepared according to Example 4. The fibrous Kraftpaper is densified according to Example 5.

The densified pellets are placed in a glass ampoule having a maximumcapacity of 250 mL. The glass ampoule is sealed under an atmosphere ofair. The pellets in the ampoule are irradiated with gamma radiation forabout 3 hours at a dose rate of about 1 Mrad per hour to provide anirradiated material in which the cellulose has a lower molecular weightthan the fibrous Kraft starting material.

Example 9 Electron Beam Processing

Samples were treated with electron beam using a vaulted Rhodotron® TT200continuous wave accelerator delivering 5 MeV electrons at 80 kW ofoutput power. Table 1 describes the parameters used. Table 2 reports thenominal dose used for the Sample ID (in MRad) and the corresponding dosedelivered to the sample (in kgy).

TABLE 1 Rhodotron ® TT 200 Parameters Beam Beam Produced: Acceleratedelectrons Beam energy: Nominal (fixed): 10 MeV (+0 keV-250 keV Energydispersion at Full width half maximum 10 Mev: (FWHM) 300 keV Beam powerat 10 MeV: Guaranteed Operating Range 1 to 80 kW Power ConsumptionStand-by condition (vacuum and <15 kW cooling ON): At 50 kW beam power:<210 kW At 80 kW beam power: <260 kW RF System Frequency: 107.5 ± 1 MHzTetrode type: Thomson TH781 Scanning Horn Nominal Scanning Length 120 cm(measured at 25-35 cm from window): Scanning Range: From 30% to 100% ofNominal Scanning Length Nominal Scanning Frequency 100 Hz ± 5% (at max.scanning length): Scanning Uniformity (across 90% ±5% of NominalScanning Length)

TABLE 2 Dosages Delivered to Samples Total Dosage (MRad) (NumberAssociated with Sample ID Delivered Dose (kgy)¹ 1 9.9 3 29.0 5 50.4 769.2 10 100.0 15 150.3 20 198.3 30 330.9 50 529.0 70 695.9 100 993.6¹For example, 9.9 kgy was delivered in 11 seconds at a beam current of 5mA and a line speed of 12.9 feet/minute. Cool time between treatmentswas around 2 minutes.

Example 10 Methods of Determining Molecular Weight of Cellulosic andLignocellulosic Materials by Gel Permeation Chromatography

Cellulosic and lignocellulosic materials for analysis were treatedaccording to Example 4. Sample materials presented in the followingtables include Kraft paper (P), wheat straw (WS), alfalfa (A), cellulose(C), switchgrass (SG), grasses (G), and starch (ST), and sucrose (S).The number “132” of the Sample ID refers to the particle size of thematerial after shearing through a 1/32 inch screen. The number after thedash refers to the dosage of radiation (MRad) and “US” refers toultrasonic treatment. For example, a sample ID “P132-10” refers to Kraftpaper that has been sheared to a particle size of 132 mesh and has beenirradiated with 10 MRad.

For samples that were irradiated with e-beam, the number following thedash refers to the amount of energy delivered to the sample. Forexample, a sample ID “P-100e” refers to Kraft paper that has beendelivered a dose of energy of about 100 MRad or about 1000 kgy (Table2).

TABLE 3 Peak Average Molecular Weight of Irradiated Kraft Paper SampleDosage¹ Average MW ± Sample Source ID (MRad) Ultrasound² Std Dev. KraftPaper P132 0 No 32853 ± 10006 P132-10 10 ″  61398 ± 2468** P132-100 100″ 8444 ± 580  P132-181 181 ″ 6668 ± 77  P132-US 0 Yes 3095 ± 1013 **Lowdoses of radiation appear to increase the molecular weight of somematerials ¹Dosage Rate = 1 MRad/hour ²Treatment for 30 minutes with 20kHz ultrasound using a 1000 W horn under re-circulating conditions withthe material dispersed in water.

TABLE 4 Peak Average Molecular Weight of Irradiated Kraft Paper withE-Beam Dosage Sample Source Sample ID (MRad) Average MW ± Std Dev. KraftPaper P-1e 1 63489 ± 595 P-5e 5 56587 ± 536 P-10e 10 53610 ± 327 P-30e30 38231 ± 124 P-70e 70 12011 ± 158 P-100e 100 9770 ± 2 

TABLE 5 Peak Average Molecular Weight of Gamma Irradiated MaterialsDosage¹ Average Sample ID Peak # (MRad) Ultrasound² MW ± Std Dev. WS1321  0 No 1407411 ± 175191 2 ″ ″ 39145 ± 3425 3 ″ ″ 2886 ± 177 WS132-10* 1 10 ″ 26040 ± 3240 WS132-100* 1 100 ″ 23620 ± 453  A132 1  0 ″ 1604886 ±151701 2 ″ ″ 37525 ± 3751 3 ″ ″ 2853 ± 490 A132-10* 1  10 ″ 50853 ± 16652 ″ ″ 2461 ± 17  A132-100* 1 100 ″ 38291 ± 2235 2 ″ ″ 2487 ± 15  SG132 1 0 ″ 1557360 ± 83693  2 ″ ″ 42594 ± 4414 3 ″ ″ 3268 ± 249 SG132-10* 1 10 ″ 60888 ± 9131 SG132-100* 1 100 ″ 22345 ± 3797 SG132-10-US 1  10 Yes 86086 ± 43518 2 ″ ″ 2247 ± 468 SG132-100-US 1 100 ″  4696 ± 1465 *Peakscoalesce after treatment **Low doses of radiation appear to increase themolecular weight of some materials ¹Dosage Rate = 1 MRad/hour ²Treatmentfor 30 minutes with 20 kHz ultrasound using a 1000 W horn underre-circulating conditions with the material dispersed in water.

TABLE 6 Peak Average Molecular Weight of Irradiated Material with E-BeamAverage MW ± STD Sample ID Peak # Dosage DEV. A-1e 1 1 1004783 ± 97518 234499 ± 482 3 2235 ± 1  A-5e 1 5 38245 ± 346 2 2286 ± 35 A-10e 1 1044326 ± 33  2 2333 ± 18 A-30e 1 30 47366 ± 583 2 2377 ± 7  A-50e 1 5032761 ± 168 2 2435 ± 6  G-1e 1 1  447362 ± 38817 2 32165 ± 779 3 3004 ±25 G-5e 1 5  62167 ± 6418 2 2444 ± 33 G-10e 1 10  72636 ± 4075 2 3065 ±34 G-30e 1 30 17159 ± 390 G-50e 1 50 18960 ± 142 ST 1 0 923336 ± 1883 2150265 ± 4033 ST-1e 1 1 846081 ± 5180 2 131222 ± 1687 ST-5e 1 5  90664 ±1370 ST-10e 1 10 98050 ± 255 ST-30e 1 30 41884 ± 223 ST-70e 1 70 9699 ±31 ST-100e 1 100 8705 ± 38

Peak average molecular weights were measured for samples treated witheither sodium bicarbonate (SBC) or tetrabutylammonium fluoride hydrate(TBAF). None of the samples reported in Table 6C showed any hydrolysis(a drop in average molecular weight.

TABLE 6C Peak Average Molecular Weights (Mp) of Treated Samples SampleID Peak # No-treatment SBC treated TBAF treated A-10e 1 53618 ± 48453271 ± 503 52995 ± 832 2 2342 ± 4  2342 ± 1  2342 ± 12 A-50e 1 33011 ±120 34469 ± 53  34830 ± 49  2 2443 ± 6  2500 ± 6  2529 ± 8  G-10e 147693 ± 173 48154 ± 535  51850 ± 1972 2 2354 ± 1  2408 ± 5  2481 ± 5 G-50e 1 33715 ± 33  35072 ± 78  32731 ± 64  P-30e 1 30313 ± 390 32809 ±54  33000 ± 69  P-70e 1 14581 ± 134 15797 ± 12  15898 ± 161 P-100e 112448 ± 28  13242 ± 2  13472 ± 3 

Gel Permeation Chromatography (GPC) is used to determine the molecularweight distribution of polymers. During GPC analysis, a solution of thepolymer sample is passed through a column packed with a porous geltrapping small molecules. The sample is separated based on molecularsize with larger molecules eluting sooner than smaller molecules. Theretention time of each component is most often detected by refractiveindex (RI), evaporative light scattering (ELS), or ultraviolet (UV) andcompared to a calibration curve. The resulting data is then used tocalculate the molecular weight distribution for the sample.

A distribution of molecular weights rather than a unique molecularweight is used to characterize synthetic polymers. To characterize thisdistribution, statistical averages are utilized. The most common ofthese averages are the “number average molecular weight” (M_(n)) and the“weight average molecular weight” (M_(w)). Methods of calculating thesevalues are described in Example 9 of PCT/US/2007/022719.

The polydispersity index or PI is defined as the ratio of M_(w)/M_(n).The larger the PI, the broader or more disperse the distribution. Thelowest value that a PI can be is 1. This represents a monodispersesample; that is, a polymer with all of the molecules in the distributionbeing the same molecular weight.

The peak molecular weight value (M_(P)) is another descriptor defined asthe mode of the molecular weight distribution. It signifies themolecular weight that is most abundant in the distribution. This valuealso gives insight to the molecular weight distribution.

Most GPC measurements are made relative to a different polymer standard.The accuracy of the results depends on how closely the characteristicsof the polymer being analyzed match those of the standard used. Theexpected error in reproducibility between different series ofdeterminations, calibrated separately, is ca. 5-10% and ischaracteristic to the limited precision of GPC determinations.Therefore, GPC results are most useful when a comparison between themolecular weight distributions of different samples is made during thesame series of determinations.

The lignocellulosic samples required sample preparation prior to GPCanalysis. First, a saturated solution (8.4% by weight) of lithiumchloride (LiCl) was prepared in dimethyl acetamide (DMAc). Approximately100 mg of the sample was added to approximately 10 g of a freshlyprepared saturated LiCl/DMAc solution, and each mixture was heated toapproximately 150° C.-170° C. with stirring for 1 hour. The resultingsolutions were generally light- to dark-yellow in color. The temperatureof the solutions was decreased to approximately 100° C. and thesolutions were heated for an additional 2 hours. The temperature of thesolutions was then decreased to approximately 50° C. and the samplesolutions were heated for approximately 48 to 60 hours. Of note, samplesirradiated at 100 Mrad were more easily solubilized as compared to theiruntreated counterpart. Additionally, the sheared samples (denoted by thenumber 132) had slightly lower average molecular weights as comparedwith uncut samples.

The resulting sample solutions were diluted 1:1 using DMAc as solventand were filtered through a 0.45 μm PTFE filter. The filtered samplesolutions were then analyzed by GPC using the parameters described inTable 7. The peak average molecular weights (Mp) of the samples, asdetermined by Gel Permeation Chromatography (GPC), are summarized inTables 3-6. Each sample was prepared in duplicate and each preparationof the sample was analyzed in duplicate (two injections) for a total offour injections per sample. The EASICAL® polystyrene standards PS1A andPS1B were used to generate a calibration curve for the molecular weightscale from about 580 to 7,500,00 Daltons.

TABLE 7 GPC Analysis Conditions Instrument: Waters Alliance GPC 2000Columns (3): PIgel 10μ Mixed-B S/N's: 10M-MB-148-83; 10M-MB- 148-84;10M-MB-174-129 Mobile Phase (solvent): 0.5% LiCl in DMAc (1.0 mL/min.)Column/Detector Temperature: 70° C. Injector Temperature: 70° C. SampleLoop Size: 323.5 μL

Example 11 Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS)Surface Analysis

Time-of-Flight Secondary Ion Mass Spectroscopy (ToF-SIMS) is asurface-sensitive spectroscopy that uses a pulsed ion beam (Cs ormicrofocused Ga) to remove molecules from the very outermost surface ofthe sample. The particles are removed from atomic monolayers on thesurface (secondary ions). These particles are then accelerated into a“flight tube” and their mass is determined by measuring the exact timeat which they reach the detector (i.e. time-of-flight). ToF-SIMSprovides detailed elemental and molecular information about the surface,thin layers, interfaces of the sample, and gives a fullthree-dimensional analysis. The use is widespread, includingsemiconductors, polymers, paint, coatings, glass, paper, metals,ceramics, biomaterials, pharmaceuticals and organic tissue. SinceToF-SIMS is a survey technique, all the elements in the periodic table,including H, are detected. ToF-SIMS data is presented in Tables 8-11.Parameters used are reported in Table 12.

TABLE 8 Normalized Mean Intensities of Various Positive Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z species Mean σ Mean σ Mean σ 23 Na 257 28 276 54 193 36 27 Al 647 43821 399 297 44 28 Si 76 45.9 197 89 81.7 10.7 15 CH₃ 77.9 7.8 161 26 13312 27 C₂H₃ 448 28 720 65 718 82 39 C₃H₃ 333 10 463 37 474 26 41 C₃H₅ 70319 820 127 900 63 43 C₃H₇ 657 11 757 162 924 118 115 C₉H₇ 73 13.4 40.34.5 42.5 15.7 128 C₁₀H₈ 55.5 11.6 26.8 4.8 27.7 6.9 73 C₃H₉Si* 181 7765.1 18.4 81.7 7.5 147 C₅H₁₅OSi₂* 72.2 33.1 24.9 10.9 38.5 4 207C₅H₁₅O₃Si₃* 17.2 7.8 6.26 3.05 7.49 1.77 647 C₄₂H₆₄PO₃ 3.63 1.05 1.431.41 10.7 7.2

TABLE 9 Normalized Mean Intensities of Various Negative Ions of Interest(Normalized relative to total ion counts × 10000) P132 P132-10 P132-100m/z species Mean σ Mean σ Mean σ 19 F 15.3 2.1 42.4 37.8 19.2 1.9 35 Cl63.8 2.8 107 33 74.1 5.5 13 CH 1900 91 1970 26 1500 6 25 C₂H 247 127 22099 540 7 26 CN 18.1 2.1 48.6 30.8 43.9 1.4 42 CNO 1.16 0.71 0.743 0.71110.8 0.9 46 NO₂ 1.87 0.38 1.66 1.65 12.8 1.8

TABLE 10 Normalized Mean Intensities of Various Positive Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 23 Na 232 56 370 37 241 44 518 57 350 27 542 104 27 Al 549 194677 86 752 371 761 158 516 159 622 166 28 Si 87.3 11.3 134 24 159 100158 32 93.7 17.1 124 11 15 CH₃ 114 23 92.9 3.9 128 18 110 16 147 16 1415 27 C₂H₃ 501 205 551 59 645 165 597 152 707 94 600 55 39 C₃H₃ 375 80288 8 379 82 321 57 435 61 417 32 41 C₃H₅ 716 123 610 24 727 182 607 93799 112 707 84 43 C₃H₇ 717 121 628 52 653 172 660 89 861 113 743 73 115C₉H₇ 49.9 14.6 43.8 2.6 42.2 7.9 41.4 10.1 27.7 8 32.4 10.5 128 C₁₀H₈38.8 13.1 39.2 1.9 35.2 11.8 31.9 7.8 21.2 6.1 24.2 6.8 73 C₃H₉Si* 92.53.0 80.6 2.9 72.3 7.7 75.3 11.4 63 3.4 55.8 2.1 147 C₅H₁₅OSi₂* 27.2 3.917.3 1.2 20.4 4.3 16.1 1.9 21.7 3.1 16.3 1.7 207 C₅H₁₅O₃Si₃* 6.05 0.743.71 0.18 4.51 0.55 3.54 0.37 5.31 0.59 4.08 0.28 647 C₄₂H₆₄PO₃ 1.611.65 1.09 1.30 0.325 0.307 nd ~ 0.868 1.31 0.306 0.334

TABLE 11 Normalized Mean Intensities of Various Negative Ions ofInterest (Normalized relative to total ion counts × 10000) P-1e P-5eP-10e P-30e P-70e P-100e m/z species Mean σ Mean σ Mean σ Mean σ Mean σMean σ 13 CH 1950 72 1700 65 1870 91 1880 35 2000 46 2120 102 25 C₂H 15447 98.8 36.3 157 4 230 17 239 22 224 19 19 F 25.4 1 24.3 1.4 74.3 18.640.6 14.9 25.6 1.9 21.5 2 35 Cl 39.2 13.5 38.7 3.5 46.7 5.4 67.6 6.245.1 2.9 32.9 10.2 26 CN 71.9 18.9 6.23 2.61 28.1 10.1 34.2 29.2 57.328.9 112 60 42 CNO 0.572 0.183 0.313 0.077 0.62 0.199 1.29 0.2 1.37 0.551.38 0.28 46 NO₂ 0.331 0.057 0.596 0.255 0.668 0.149 1.44 0.19 1.92 0.290.549 0.1

TABLE 12 ToF-SIMS Parameters Instrument Conditions: Instrument: PHITRIFT II Primary Ion Source: ⁶⁹Ga Primary Ion Beam Potential: 12 kV +ions 18 kV − ions Primary Ion Current (DC): 2 na for P#E samples 600 pAfor P132 samples Energy Filter/CD: Out/Out Masses Blanked: None ChargeCompensation: On

ToF-SIMS uses a focused, pulsed particle beam (typically Cs or Ga) todislodge chemical species on a materials surface. Particles producedcloser to the site of impact tend to be dissociated ions (positive ornegative). Secondary particles generated farther from the impact sitetend to be molecular compounds, typically fragments of much largerorganic macromolecules. The particles are then accelerated into a flightpath on their way towards a detector. Because it is possible to measurethe “time-of-flight” of the particles from the time of impact todetector on a scale of nano-seconds, it is possible to produce a massresolution as fine as 0.00× atomic mass units (i.e. one part in athousand of the mass of a proton). Under typical operating conditions,the results of ToF-SIMS analysis include: a mass spectrum that surveysall atomic masses over a range of 0-10,000 amu, the rastered beamproduces maps of any mass of interest on a sub-micron scale, and depthprofiles are produced by removal of surface layers by sputtering underthe ion beam. Negative ion analysis showed that the polymer hadincreasing amounts of CNO, CN, and NO₂ groups.

Example 12 X-Ray Photoelectron Spectroscopy (XPS)/Electron Spectroscopyfor Chemical Analysis (ESCA)

X-Ray Photoelectron Spectroscopy (XPS) (sometimes called “ESCA”)measures the chemical composition of the top five nanometers of surface;XPS uses photo-ionization energy and energy-dispersive analysis of theemitted photoelectrons to study the composition and electronic state ofthe surface region of a sample. X-ray Photoelectron spectroscopy isbased upon a single photon in/electron out. Soft x-rays stimulate theejection of photoelectrons whose kinetic energy is measured by anelectrostatic electron energy analyzer. Small changes to the energy arecaused by chemically-shifted valence states of the atoms from which theelectrons are ejected; thus, the measurement provides chemicalinformation about the sample surface.

TABLE 13 Atomic Concentrations (in %)^(a,b) Atom Sample ID C O Al SiP132 (Area1) 57.3 39.8 1.5 1.5 P132 (Area2) 57.1 39.8 1.6 1.5 P132-10(Area 1) 63.2 33.5 1.7 1.6 P132-10 (Area 2) 65.6 31.1 1.7 1.7 P132-100(Area 1) 61.2 36.7 0.9 1.2 P132-100 (Area 2) 61 36.9 0.8 1.3^(a)Normalized to 100% of the elements detected. XPS does not detect Hor He.

TABLE 14 Carbon Chemical State (in % C) Sample ID C—C, C—H C—O C═O O—C═OP132 (Area1) 22 49 21 7 P132 (Area2) 25 49 20 6 P132-10 (Area 1) 34 4215 9 P132-10 (Area 2) 43 38 14 5 P132-100 (Area 1) 27 45 15 9 P132-100(Area 2) 25 44 23 9

TABLE 15 Atomic Concentrations (in %)^(a,b) Atom Sample ID C O Al Si NaP-1e (Area 1) 59.8 37.9 1.4 0.9 ~ P-1e (Area 2) 58.5 38.7 1.5 1.3 ~ P-5e(Area 1) 58.1 39.7 1.4 0.8 ~ P-5e (Area 2) 58.0 39.7 1.5 0.8 ~ P-10e(Area 1) 61.6 36.7 1.1 0.7 ~ P-10e (Area 2) 58.8 38.6 1.5 1.1 ~ P-50e(Area 1) 59.9 37.9 1.4 0.8 <0.1 P-50e (Area 2) 59.4 38.3 1.4 0.9 <0.1P-70e (Area 1) 61.3 36.9 1.2 0.6 <0.1 P-70e (Area 2) 61.2 36.8 1.4 0.7<0.1 P-100e (Area 1) 61.1 37.0 1.2 0.7 <0.1 P-100e (Area 2) 60.5 37.21.4 0.9 <0.1 ^(a)Normalized to 100% of the elements detected. XPS doesnot detect H or He. ^(b)A less than symbol “<” indicates accuratequantification cannot be made due to weak signal intensity.

TABLE 16 Carbon Chemical State Table (in % C) Sample ID C—C, C—H C—O C═OO—C═O P-1e (Area 1) 29 46 20 5 P-1e (Area 2) 27 49 19 5 P-5e (Area 1) 2553 18 5 P-5e (Area 2) 28 52 17 4 P-10e (Area 1) 33 47 16 5 P-10e (Area2) 28 51 16 5 P-50e (Area 1) 29 45 20 6 P-50e (Area 2) 28 50 16 5 P-70e(Area 1) 32 45 16 6 P-70e (Area 2) 35 43 16 6 P-100e (Area 1) 32 42 19 7P-100e (Area 2) 30 47 16 7

TABLE 17 Analytical Parameters Instrument: PHI Quantum 2000 X-raysource: Monochromated Alk_(α) 1486.6 eV Acceptance Angle: ±23° Take-offangle: 45° Analysis area: 1400 × 300 μm Charge Correction: C1s 284.8 eV

XPS spectra are obtained by irradiating a material with a beam ofaluminum or magnesium X-rays while simultaneously measuring the kineticenergy (KE) and number of electrons that escape from the top 1 to 10 nmof the material being analyzed (see analytical parameters, Table 17).The XPS technique is highly surface specific due to the short range ofthe photoelectrons that are excited from the solid. The energy of thephotoelectrons leaving the sample is determined using a ConcentricHemispherical Analyzer (CHA) and this gives a spectrum with a series ofphotoelectron peaks. The binding energy of the peaks is characteristicof each element. The peak areas can be used (with appropriatesensitivity factors) to determine the composition of the materialssurface. The shape of each peak and the binding energy can be slightlyaltered by the chemical state of the emitting atom. Hence XPS canprovide chemical bonding information as well. XPS is not sensitive tohydrogen or helium, but can detect all other elements. XPS requiresultra-high vacuum (UHV) conditions and is commonly used for the surfaceanalysis of polymers, coatings, catalysts, composites, fibers, ceramics,pharmaceutical/medical materials, and materials of biological origin.XPS data is reported in Tables 13-16.

Example 13 Raman Analysis

Raman spectra were acquired from the surface of fibers from samples:P132, P132-100, P-1e, and P-100e. The measurements were performed usinga “LabRam” J-Y Spectrometer. A HeNe laser (632.8 nm wavelength) and 600gr/mm grating were used for the measurements. The measurements wereperformed confocally using backscattering geometry (180°) under anOlympus BX40 microscope. The samples had a Raman spectrum typical ofcellulose.

Example 14 Scanning Probe Microscopy (SPM) Surface Analysis Using anAtomic Force Microscope (AFM)

The purpose of this analysis was to obtain Atomic Force Microscope (AFM)images of the samples in Tables 18 and 19 to measure surface roughness.

Scanning probe microscopy (SPM) is a branch of microscopy that formsimages of surfaces using a physical probe that scans the specimen. Animage of the surface is obtained by mechanically moving the probe in araster scan of the specimen, line by line, and recording theprobe-surface interaction as a function of position. The atomic forcemicroscope (AFM) or scanning force microscope (SFM) is a veryhigh-resolution type of scanning probe microscope, with demonstratedresolution of fractions of a nanometer, more than 1000 times better thanthe optical diffraction limit. The probe (or the sample under astationary probe) generally is moved by a piezoelectric tube. Suchscanners are designed to be moved precisely in any of the threeperpendicular axes (x,y,z). By following a raster pattern, the sensordata forms an image of the probe-surface interaction. Feedback from thesensor is used to maintain the probe at a constant force or distancefrom the object surface. For atomic force microscopy, the sensor is aposition-sensitive photodetector that records the angle of reflectionfrom a laser bean focused on the top of the cantilever.

TABLE 18 Roughness Results for Gamma-Irradiated Samples Sample ID RMS(Å) R_(a) (Å) R_(max) (Å) P132 927.2 716.3 8347.6 P132-10 825.7 576.811500 P132-100 1008 813.5 7250.7

TABLE 19 Roughness Results for Samples Irradiated with E-Beam Sample IDRMS (Å) R_(a) (Å) R_(max) (Å) P-1e 1441.2 1147.1 8955.4 P-5e 917.3 727.56753.4 P-10e 805.6 612.1 7906.5 P-30e 919.2 733.7 6900 P-70e 505.8 388.15974.2 P-100e 458.2 367.9 3196.9

AFM images were collected using a NanoScope III Dimension 5000 (DigitalInstruments, Santa Barbara, Calif., USA). The instrument was calibratedagainst a NIST traceable standard with an accuracy better than 2%.NanoProbe silicon tips were used. Image processing procedures involvingauto-flattening, plane fitting or convolution were employed.

One 5 μm×5 μm area was imaged at a random location on top of a singlefiber. Perspective (3-D) views of these surfaces are included withvertical exaggerations noted on the plots (FIGS. 31A-31F). The roughnessanalyses were performed and are expressed in: (1) Root-Mean-SquareRoughness, RMS; (2) Mean Roughness, Ra; and (3) Maximum Height(Peak-to-Valley), Rmax. Results are summarized in Tables 18 and 19.

Example 15 Determining Crystallinity of Irradiated Materials by X-RayDiffraction

X-ray diffraction (XRD) is a method by which a crystalline sample isirradiated with monoenergetic x-rays. The interaction of the latticestructure of the sample with these x-rays is recorded and providesinformation about the crystalline structure being irradiated. Theresulting characteristic “fingerprint” allows for the identification ofthe crystalline compounds present in the sample. Using a whole-patternfitting analysis (the Rietvelt Refinement), it is possible to performquantitative analyses on samples containing more than one crystallinecompound.

TABLE 20 XRD Data Including Domain Size and % Crystallinity Domain SizeSample ID (Å) % Crystallinity P132 55 55 P132-10 46 58 P132-100 50 55P132-181 48 52 P132-US 26 40 A132 28 42 A132-10 26 40 A132-100 28 35WS132 30 36 WS132-10 27 37 WS132-100 30 41 SG132 29 40 SG132-10 28 38SG132-100 28 37 SG132-10-US 25 42 SG132-100-US 21 34

Each sample was placed on a zero background holder and placed in aPhillips PW1800 diffractometer using Cu radiation. Scans were then runover the range of 5° to 50° with a step size of 0.05° and a countingtime of 2 hours each.

Once the diffraction patterns were obtained, the phases were identifiedwith the aid of the Powder Diffraction File published by theInternational Centre for Diffraction Data. In all samples thecrystalline phase identified was cellulose—Ia, which has a triclinicstructure.

The distinguishing feature among the 20 samples is the peak breadth,which is related to the crystallite domain size. The experimental peakbreadth was used to compute the domain size and percent crystallinity,which are reported in Table 4.

Percent crystallinity (X_(c)%) is measured as a ratio of the crystallinearea to the total area under the x-ray diffraction peaks and equals100%×(A_(c)/(A_(a)+A_(c)), where

A_(c) = Area of crystalline phase A_(a) = Area of amorphous phase X_(c)= Percent of crystallinity

To determine the percent crystallinity for each sample it was necessaryto first extract the amount of the amorphous phase. This is done byestimating the area of each diffraction pattern that can be attributedto the crystalline phase (represented by the sharper peaks) and thenon-crystalline phase (represented by the broad humps beneath thepattern and centered at 22° and 38°).

A systematic process was used to minimize error in these calculationsdue to broad crystalline peaks as well as high background intensity,First, a linear background was applied and then removed. Second, twoGaussian peaks centered at 22° and 38° with widths of 10-12° each werefitted to the humps beneath the crystalline peaks. Third, the areabeneath the two broad Gaussian peaks and the rest of the pattern weredetermined. Finally, percent crystallinity was calculated by dividingthe area beneath the crystalline peak by the total intensity (afterbackground subtraction). Domain size and % crystallinity of the samplesas determined by X-ray diffraction (XRD) are presented in Table 20.

Example 16 Porosimetry Analysis of Irradiated Materials

Mercury pore size and pore volume analysis (Table 21) is based onforcing mercury (a non-wetting liquid) into a porous structure undertightly controlled pressures. Since mercury does not wet most substancesand will not spontaneously penetrate pores by capillary action, it mustbe forced into the voids of the sample by applying external pressure.The pressure required to fill the voids is inversely proportional to thesize of the pores. Only a small amount of force or pressure is requiredto fill large voids, whereas much greater pressure is required to fillvoids of very small pores.

TABLE 21 Pore Size and Volume Distribution by Mercury Porosimetry MedianMedian Average Bulk Total Total Pore Pore Pore Density ApparentIntrusion Pore Diameter Diameter Diameter @ 0.50 (skeletal) Volume Area(Volume) (Area) (4V/A) psia Density Porosity Sample ID (mL/g) (m²/g)(μm) (μm) (μm) (g/mL) (g/mL) (%) P132 6.0594 1.228 36.2250 13.727819.7415 0.1448 1.1785 87.7163 P132-10 5.5436 1.211 46.3463 4.564618.3106 0.1614 1.5355 89.4875 P132-100 5.3985 0.998 34.5235 18.200521.6422 0.1612 1.2413 87.0151 P132-181 3.2866 0.868 25.3448 12.241015.1509 0.2497 1.3916 82.0577 P132-US 6.0005 14.787 98.3459 0.00551.6231 0.1404 0.8894 84.2199 A132 2.0037 11.759 64.6308 0.0113 0.68160.3683 1.4058 73.7990 A132-10 1.9514 10.326 53.2706 0.0105 0.7560 0.37681.4231 73.5241 A132-100 1.9394 10.205 43.8966 0.0118 0.7602 0.37601.3889 72.9264 SG132 2.5267 8.265 57.6958 0.0141 1.2229 0.3119 1.470878.7961 SG132-10 2.1414 8.643 26.4666 0.0103 0.9910 0.3457 1.331574.0340 SG132-100 2.5142 10.766 32.7118 0.0098 0.9342 0.3077 1.359077.3593 SG132-10-US 4.4043 1.722 71.5734 1.1016 10.2319 0.1930 1.288385.0169 SG132-100-US 4.9665 7.358 24.8462 0.0089 2.6998 0.1695 1.073184.2010 WS132 2.9920 5.447 76.3675 0.0516 2.1971 0.2773 1.6279 82.9664WS132-10 3.1138 2.901 57.4727 0.3630 4.2940 0.2763 1.9808 86.0484WS132-100 3.2077 3.114 52.3284 0.2876 4.1199 0.2599 1.5611 83.3538 A-1e1.9535 3.698 25.3411 0.0810 2.1130 0.3896 1.6299 76.0992 A-5e 1.96976.503 29.5954 0.0336 1.2117 0.3748 1.4317 73.8225 A-10e 2.0897 12.03045.5493 0.0101 0.6948 0.3587 1.4321 74.9545 A-50e 2.1141 7.291 37.07600.0304 1.1599 0.3577 1.4677 75.6264 G-1e 2.4382 7.582 58.5521 0.02011.2863 0.3144 1.3472 76.6610 G-5e 2.4268 6.436 44.4848 0.0225 1.50820.3172 1.3782 76.9831 G-10e 2.6708 6.865 62.8605 0.0404 1.5562 0.29601.4140 79.0638 G-50e 2.8197 6.798 56.5048 0.0315 1.6591 0.2794 1.317978.7959 P-1e 7.7692 1.052 49.8844 22.9315 29.5348 0.1188 1.5443 92.3065P-5e 7.1261 1.212 46.6400 12.3252 23.5166 0.1268 1.3160 90.3644 P-10e6.6096 1.113 41.4252 17.4375 23.7513 0.1374 1.4906 90.7850 P-50e 6.59111.156 40.7837 15.9823 22.7974 0.1362 1.3302 89.7616 P-100e 5.3507 1.19535.3622 10.7400 17.9063 0.1648 1.3948 88.1840 S 0.4362 0.030 102.841142.5047 57.8208 0.9334 1.5745 40.7160 S-1e 0.3900 0.632 90.6808 0.00412.4680 0.9772 1.5790 38.1140 S-5e 0.3914 0.337 97.1991 0.0070 4.64060.9858 1.6052 38.5847 S-10e 0.4179 0.349 113.4360 0.0042 4.7873 0.94691.5669 39.5678 S-30e 0.4616 5.329 102.0559 0.0042 0.3464 0.9065 1.558541.8388 S-50e 0.5217 7.162 137.2124 0.0051 0.2914 0.8521 1.5342 44.4582S-100e 0.8817 15.217 76.4577 0.0053 0.2318 0.6478 1.5105 57.1131 St0.6593 17.631 4.2402 0.0053 0.1496 0.7757 1.5877 51.1438 St-1e 0.672018.078 4.3360 0.0052 0.1487 0.7651 1.5750 51.4206 St-5e 0.6334 19.4954.2848 0.0051 0.1300 0.7794 1.5395 49.3706 St-10e 0.6208 16.980 4.33620.0056 0.1462 0.7952 1.5703 49.3630 St-30e 0.6892 18.066 4.4152 0.00500.1526 0.7475 1.5417 51.5165 St-50e 0.6662 18.338 4.3759 0.0054 0.14530.7637 1.5548 50.8778 St-100e 0.6471 23.154 5.4032 0.0048 0.1118 0.72291.3582 46.7761

The AutoPore 9520 can attain a maximum pressure of 414 MPa or 60,000psia. There are four low pressure stations for sample preparation andcollection of macropore data from 0.2 psia to 50 psia. There are twohigh pressure chambers, which collect data from 25 psia to 60,000 psia.The sample is placed in a bowl-like apparatus called a penetrometer,which is bonded to a glass capillary stem with a metal coating. Asmercury invades the voids in and around the sample, it moves down thecapillary stem. The loss of mercury from the capillary stem results in achange in the electrical capacitance. The change in capacitance duringthe experiment is converted to volume of mercury by knowing the stemvolume of the penetrometer in use. A variety of penetrometers withdifferent bowl (sample) sizes and capillaries are available toaccommodate most sample sizes and configurations. Table 22 below definessome of the key parameters calculated for each sample.

TABLE 22 Definition of Parameters Parameter Description Total IntrusionVolume: The total volume of mercury intruded during an experiment. Thiscan include interstitial filling between small particles, porosity ofsample, and compression volume of sample. Total Pore Area: The totalintrusion volume converted to an area assuming cylindrical shaped pores.Median Pore Diameter (volume): The size at the 50^(th) percentile on thecumulative volume graph. Median Pore Diameter (area): The size at the50^(th) percentile on the cumulative area graph. Average Pore Diameter:The total pore volume divided by the total pore area (4 V/A). BulkDensity: The mass of the sample divided by the bulk volume. Bulk volumeis determined at the filling pressure, typically 0.5 psia. ApparentDensity: The mass of sample divided by the volume of sample measured athighest pressure, typically 60,000 psia. Porosity: (BulkDensity/Apparent Density) × 100%

Example 17 Particle Size Analysis of Irradiated Materials

The technique of particle sizing by static light scattering is based onMie theory (which also encompasses Fraunhofer theory). Mie theorypredicts the intensity vs. angle relationship as a function of the sizefor spherical scattering particles provided that other system variablesare known and held constant. These variables are the wavelength ofincident light and the relative refractive index of the sample material.Application of Mie theory provides the detailed particle sizeinformation. Table 23 summarizes particle size using median diameter,mean diameter, and modal diameter as parameters.

TABLE 23 Particle Size by Laser Light Scattering (Dry Sample Dispersion)Median Mean Modal Sample ID Diameter (μm) Diameter (μm) Diameter (μm)A132 380.695 418.778 442.258 A132-10 321.742 366.231 410.156 A132-100301.786 348.633 444.169 SG132 369.400 411.790 455.508 SG132-10 278.793325.497 426.717 SG132-100 242.757 298.686 390.097 WS132 407.335 445.618467.978 WS132-10 194.237 226.604 297.941 WS132-100 201.975 236.037307.304

Particle size was determined by Laser Light Scattering (Dry SampleDispersion) using a Malvern Mastersizer 2000 using the followingconditions:

Feed Rate: 35% Disperser Pressure: 4 Bar Optical Model (2.610, 1.000i),1.000

An appropriate amount of sample was introduced onto a vibratory tray.The feed rate and air pressure were adjusted to ensure that theparticles were properly dispersed. The key component is selecting an airpressure that will break up agglomerations, but does not compromise thesample integrity. The amount of sample needed varies depending on thesize of the particles. In general, samples with fine particles requireless material than samples with coarse particles.

Example 18 Surface Area Analysis of Irradiated Materials

Surface area of each sample was analyzed using a Micromeritics ASAP 2420Accelerated Surface Area and Porosimetry System. The samples wereprepared by first degassing for 16 hours at 40° C. Next, free space(both warm and cold) with helium is calculated and then the sample tubeis evacuated again to remove the helium. Data collection begins afterthis second evacuation and consists of defining target pressures whichcontrols how much gas is dosed onto the sample. At each target pressure,the quantity of gas adsorbed and the actual pressure are determined andrecorded. The pressure inside the sample tube is measured with apressure transducer. Additional doses of gas will continue until thetarget pressure is achieved and allowed to equilibrate. The quantity ofgas adsorbed is determined by summing multiple doses onto the sample.The pressure and quantity define a gas adsorption isotherm and are usedto calculate a number of parameters, including BET surface area (Table24).

TABLE 24 Summary of Surface Area by Gas Adsorption BET Surface Sample IDSingle point surface area (m²/g) Area (m²/g) P132 @ P/Po = 0.2503877711.5253 1.6897 P132-10 @ P/Po = 0.239496722 1.0212 1.2782 P132-100 @ P/Po= 0.240538100 1.0338 1.2622 P132-181 @ P/Po = 0.239166091 0.5102 0.6448P132-US @ P/Po = 0.217359072 1.0983 1.6793 A132 @ P/Po = 0.2400406100.5400 0.7614 A132-10 @ P/Po = 0.211218936 0.5383 0.7212 A132-100 @ P/Po= 0.238791097 0.4258 0.5538 SG132 @ P/Po = 0.237989353 0.6359 0.8350SG132-10 @ P/Po = 0.238576905 0.6794 0.8689 SG132-100 @ P/Po =0.241960361 0.5518 0.7034 SG132-10-US @ P/Po = 0.225692889 0.5693 0.7510SG132-100-US @ P/Po = 0.225935246 1.0983 1.4963 G-10-US 0.751 G100-US1.496 G132-US 1.679 WS132 @ P/Po = 0.237823664 0.6582 0.8663 WS132-10 @P/Po = 0.238612476 0.6191 0.7912 WS132-100 @ P/Po = 0.238398832 0.62550.8143 A-1e @ P/Po = 0.238098138 0.6518 0.8368 A-5e @ P/Po = 0.2431844770.6263 0.7865 A-10e @ P/Po = 0.243163236 0.4899 0.6170 A-50e @ P/Po =0.243225512 0.4489 0.5730 G-1e @ P/Po = 0.238496102 0.5489 0.7038 G-5e @P/Po = 0.242792602 0.5621 0.7086 G-10e @ P/Po = 0.243066031 0.50210.6363 G-50e @ P/Po = 0.238291132 0.4913 0.6333 P-1e @ P/Po =0.240842223 1.1413 1.4442 P-5e @ P/Po = 0.240789274 1.0187 1.3288 P-10e@ P/Po = 0.240116967 1.1015 1.3657 P-50e @ P/Po = 0.240072114 1.00891.2593 P-100e @ P/Po = 0.236541386 0.9116 1.1677 S @ P/Po = 0.2253350380.0147 0.0279 S-1e @ P/Po = 0.217142291 0.0193 0.0372 S-5e @ P/Po =0.133107838 0.0201 0.0485 S-10e @ P/Po = 0.244886517 0.0236 0.0317 S-30e@ P/Po = 0.237929400 0.0309 0.0428 S-50e @ P/Po = 0.245494494 0.02620.0365 S-100e @ P/Po = 0.224698551 0.0368 0.0506 St @ P/Po = 0.2383249490.3126 0.4013 St-1e @ P/Po = 0.238432726 0.3254 0.4223 St-5e @ P/Po =0.238363587 0.3106 0.4071 St-10e @ P/Po = 0.238341099 0.3205 0.4268St-30e @ P/Po = 0.238629889 0.3118 0.4189 St-50e @ P/Po = 0.2446309800.3119 0.3969 St-100e @ P/Po = 0.238421621 0.2932 0.3677

The BET model for isotherms is a widely used theory for calculating thespecific surface area. The analysis involves determining the monolayercapacity of the sample surface by calculating the amount required tocover the entire surface with a single densely packed layer of krypton.The monolayer capacity is multiplied by the cross sectional area of amolecule of probe gas to determine the total surface area. Specificsurface area is the surface area of the sample aliquot divided by themass of the sample.

Example 19 Fiber Length Determination of Irradiated Materials

Fiber length distribution testing was performed in triplicate on thesamples submitted using the Techpap MorFi LB01 system. The average fiberlength and width are reported in Table 25.

TABLE 25 Summary of Lignocellulosic Fiber Length and Width Data Arith-Average Statistically Width metic Length Corrected Average (micro-Average Weighted in Length Weighted meters) Sample ID (mm) Length (mm)in Length (mm) (μm) P132-10 0.484 0.615 0.773 24.7 P132-100 0.369 0.4230.496 23.8 P132-181 0.312 0.342 0.392 24.4 A132-10 0.382 0.423 0.65043.2 A132-100 0.362 0.435 0.592 29.9 SG132-10 0.328 0.363 0.521 44.0SG132-100 0.325 0.351 0.466 43.8 WS132-10 0.353 0.381 0.565 44.7WS132-100 0.354 0.371 0.536 45.4

Example 20 Ultrasonic Treatment of Irradiated and Un-IrradiatedSwitchgrass

Switchgrass was sheared according to Example 4. The switchgrass wastreated by ultrasound alone or irradiation with 10 Mrad or 100 Mrad ofgamma rays, and then sonicated. The resulting materials correspond toG132-BR (un-irradiated), G132-10-BR (10 Mrad and sonication) andG132-100-BR (100 Mrad and sonication), as presented in Table 1.Sonication was performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Eachsample was dispersed in water at a concentration of about 0.10 g/mL.

FIGS. 32 and 33 show the apparatus used for sonication. Apparatus 500includes a converter 502 connected to booster 504 communicating with ahorn 506 fabricated from titanium or an alloy of titanium. The horn,which has a seal 510 made from VITON® about its perimeter on itsprocessing side, forms a liquid tight seal with a processing cell 508.The processing side of the horn is immersed in a liquid, such as water,that has dispersed therein the sample to be sonicated. Pressure in thecell is monitored with a pressure gauge 512. In operation, each sampleis moved by pump 517 from tank 516 through the processing cell and issonicated. After, sonication, the sample is captured in tank 520. Theprocess can be reversed in that the contents of tank 520 can be sentthrough the processing cell and captured in tank 516. This process canbe repeated a number of times until a desired level of processing isdelivered to the sample.

Example 21 Scanning Electron Micrographs of Un-Irradiated Switchgrass inComparison to Irradiated and Irradiated and Sonicated Switchgrass

Switchgrass samples for the scanning electron micrographs were appliedto carbon tape and gold sputter coated (70 seconds). Images were takenwith a JEOL 6500 field emission scanning electron microscope.

FIG. 34 is a scanning electron micrograph at 1000× magnification of afibrous material produced from shearing switchgrass on a rotary knifecutter, and then passing the sheared material through a 1/32 inchscreen.

FIGS. 35 and 36 are scanning electron micrographs of the fibrousmaterial of FIG. 34 after irradiation with 10 Mrad and 100 Mrad gammarays, respectively, at 1000× magnification.

FIG. 37 is a scanning electron micrographs of the fibrous material ofFIG. 34 after irradiation with 10 Mrad and sonication at 1000×magnification.

FIG. 38 is a scanning electron micrographs of the fibrous material ofFIG. 34 after irradiation with 100 Mrad and sonication at 1000×magnification.

Example 22 Fourier Transform Infrared (FT-IR) Spectrum of Irradiated andUnirradiated Kraft Paper

FT-IR analysis was performed on a Nicolet/Impact 400. The resultsindicate that samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e,P-70e, and P-100e are consistent with a cellulose-based material.

FIG. 39 is an infrared spectrum of Kraft board paper sheared accordingto Example 4, while FIG. 40 is an infrared spectrum of the Kraft paperof FIG. 39 after irradiation with 100 Mrad of gamma radiation. Theirradiated sample shows an additional peak in region A (centered about1730 cm⁻¹) that is not found in the un-irradiated material. Of note, anincrease in the amount of a carbonyl absorption at ˜1650 cm⁻¹ wasdetected when going from P132 to P132-10 to P132-100. Similar resultswere observed for the samples P-1e, P-5e, P-10e, P-30e, P-70e, andP-100e.

FIGS. 40-1 to 40-4 are infrared spectra of alfalfa (A), alfalfairradiated at 50e (A-50e), sucrose irradiated at 50e (S-50e), andsucrose irradiated at 100e (S-100e), respectively. Of note, an increasein the amount of a carbonyl absorption at ˜1650 cm-1 was detected forsample A-50e, as well as S-100e.

The alfalfa samples showed a small peak present at 1720 cm⁻¹ in theuntreated sample, which grows to the most dominant peak in the A-50espectrum. There was no significant change in the IR spectrum forsucrose. S-100e was the only spectrum which showed two small new peaksat 1713 and 1647 cm⁻¹.

Example 23 Proton and Carbon-13 Nuclear Magnetic Resonance (¹H-NMR and¹³C-NMR) Spectra of Irradiated and Unirradiated Kraft Paper SamplePreparation

The samples P132, P132-10, P132-100, P-1e, P-5e, P-10e, P-30e, P-70e,and P-100e were prepared for analysis by dissolution with DMSO-d₆ with2% tetrabutyl ammonium fluoride trihydrate. The samples which hadundergone lower levels of irradiation were significantly less solublethan the samples with higher irradiation. Unirradiated samples formed agel in this solvent mixture, but heating to 60° C. resolved the peaks inthe NMR spectra. The samples having undergone higher levels ofirradiation were soluble at a concentration of 10% wt/wt.

Analysis ¹H-NMR spectra of the samples at 15 mg/mL showed a distinctvery broad resonance peak centered at 16 ppm (FIGS. 40A-40J). This peakis characteristic of an exchangeable —OH proton for an enol and wasconfirmed by a “D₂O shake.” Model compounds (acetylacetone, glucuronicacid, and keto-gulonic acid) were analyzed and made a convincing casethat this peak was indeed an exchangeable enol proton. This proposedenol peak was very sensitive to concentration effects, and we wereunable to conclude whether this resonance was due to an enol or possiblya carboxylic acid.

The carboxylic acid proton resonances of the model compounds weresimilar to what was observed for the treated cellulose samples. Thesemodel compounds were shifted up field to ˜5-6 ppm. Preparation of P-100eat higher concentrations (˜10% wt/wt) led to the dramatic down fieldshifting to where the carboxylic acid resonances of the model compoundswere found (˜6 ppm) (FIG. 40N). These results lead to the conclusionthat this resonance is unreliable for characterizing this functionalgroup, however the data suggests that the number of exchangeablehydrogens increases with increasing irradiation of the sample. Also, novinyl protons were detected.

The ¹³C NMR spectra of the samples confirm the presence of a carbonyl ofa carboxylic acid or a carboxylic acid derivative. This new peak (at 168ppm) is not present in the untreated samples (FIG. 40K). A ¹³C NMRspectrum with a long delay allowed the quantitation of the signal forP-100e (FIGS. 40L-40M). Comparison of the integration of the carbonylresonance to the resonances at approximately 100 ppm (the C1 signals)suggests that the ratio of the carbonyl carbon to C1 is 1:13.8 orroughly 1 carbonyl for every 14 glucose units. The chemical shift at 100ppm correlates well with glucuronic acid.

The ¹³C NMR spectrum for A-50E (166,000 scans; 38 h) shows aromaticcarbons of lignin (˜130 ppm) and also shows multiple carbonyl resonancesaround 170 ppm (the P samples showed only one resonance). The ¹H NMRclearly shows the aromatic signals from lignin.

The ¹³C NMR spectrum for S-100E does not show a carbonyl resonance at170 ppm as the other treated samples do, but the ¹³C NMR spectrum showsthat there has been extensive reaction and there are now over 40 carbonresonances in the spectrum while untreated sucrose has only 12 signals(104.71, 93.20, 82.42 77.51 75.09, 73.68, 73.44, 72.14, 70.31, 63.44,62.46, 61.24 ppm). These signals are more intense comparing the spectrafrom S-70E to S-100E. The ¹H NMR spectra have many overlapping peaks andare not easily interpreted.

Manual Titration

Samples P-100e and P132-100 (1 g) were suspended in deionized water (25mL). The indicator alizarin yellow was added to each sample withstirring. P-100e was more difficult to wet. Both samples were titratedwith a solution of 0.2M NaOH. The end point was very subtle and wasconfirmed by using pH paper. The starting pH of the samples was ˜4 forboth samples. P132-100 required 0.4 milliequivalents of hydroxide, whichgives a molecular weight for the carboxylic acid of 2500 amu. If 180 amuis used for a monomer, this suggests there is one carboxylic acid groupfor 13.9 monomer units. Likewise, P-100e required 3.2 milliequivalentsof hydroxide, which calculates to be one carboxylic acid group for every17.4 monomer units.

CONCLUSIONS

The C-6 carbon of cellulose appears to be oxidized to the carboxylicacid (a glucuronic acid derivative) in this oxidation is surprisinglyspecific. This oxidation is in agreement with the IR band that growswith irradiation at ˜1740 cm⁻¹, which corresponds to an aliphaticcarboxylic acid. The titration results are in agreement with thequantitative ¹³C NMR. The increased solubility of the sample with thehigher levels of irradiation correlates well with the increasing numberof carboxylic acid protons. A proposed mechanism for the degradation of“C-6 oxidized cellulose” is provided below in Scheme 1.

Potentiometric Titration Analysis

A potentiometer (Metrohm Ion Analysis 794 Basic Titrino) was used tomeasure the electrode potential of sample solutions and therefore anaccurate titration analysis based on a redox reaction was achieved. Thepotential of the working electrode will suddenly change as the endpointis reached.

Results

P-30E had one carboxylic acid per 57 saccharide units. P-70E had onecarboxylic acid unit per 27 saccharide units. P-100E had one carboxylicacid per 22 saccharide units. Of particular interest, the samplesdarkened significantly upon titration to a rusty red color. (This wasnot noticeable during the manual titrations). A titration curve forsample P-30e is presented in FIG. 40O.

Example 24 Combination of Electron Beam and Sonication Pretreatment

Switchgrass is used as the feedstock and is sheared with a Munson rotaryknife cutter into a fibrous material. The fibrous material is thenevenly distributed onto an open tray composed of tin with an area ofgreater than about 500 in². The fibrous material is distributed so thatit has a depth of about 1-2 inches in the open tray. The fibrousmaterial may be distributed in plastic bags at lower doses ofirradiation (under 10 Mrad), and left uncovered on the metal tray athigher doses of radiation.

Separate samples of the fibrous material are then exposed to successivedoses of electron beam radiation to achieve a total dose of 1 Mrad, 2Mrad, 3, Mrad, 5 Mrad, 10 Mrad, 50 Mrad, and 100 Mrad. Some samples aremaintained under the same conditions as the remaining samples, but arenot irradiated, to serve as controls. After cooling, the irradiatedfibrous material is sent on for further processing through a sonicationdevice.

The sonication device includes a converter connected to boostercommunicating with a horn fabricated from titanium or an alloy oftitanium. The horn, which has a seal made from VITON® about itsperimeter on its processing side, forms a liquid tight seal with aprocessing cell. The processing side of the horn is immersed in aliquid, such as water, into which the irradiated fibrous material to besonicated is immersed. Pressure in the cell is monitored with a pressuregauge. In operation, each sample is moved by pump through the processingcell and is sonicated.

To prepare the irradiated fibrous material for sonication, theirradiated fibrous material is removed from any container (e.g., plasticbags) and is dispersed in water at a concentration of about 0.10 g/mL.Sonication is performed on each sample for 30 minutes using 20 kHzultrasound from a 1000 W horn under re-circulating conditions. Aftersonication, the irradiated fibrous material is captured in a tank. Thisprocess can be repeated a number of times until a desired level ofprocessing is achieved based on monitoring the structural changes in theswitchgrass. Again, some irradiated samples are kept under the sameconditions as the remaining samples, but are not sonicated, to serve ascontrols. In addition, some samples that were not irradiated aresonicated, again to serve as controls. Thus, some controls are notprocessed, some are only irradiated, and some are only sonicated.

Example 25 Microbial Testing of Pretreated Biomass

Specific lignocellulosic materials pretreated as described herein areanalyzed for toxicity to common strains of yeast and bacteria used inthe biofuels industry for the fermentation step in ethanol production.Additionally, sugar content and compatibility with cellulase enzymes areexamined to determine the viability of the treatment process. Testing ofthe pretreated materials is carried out in two phases as follows.

Phase 1: Toxicity and Sugar Content

Toxicity of the pretreated grasses and paper feedstocks is measured inyeast Saccharomyces cerevisiae (wine yeast) and Pichia stipitis (ATCC66278) as well as the bacteria Zymomonas mobilis (ATCC 31821) andClostridium thermocellum (ATCC 31924). A growth study is performed witheach of the organisms to determine the optimal time of incubation andsampling.

Each of the feedstocks is then incubated, in duplicate, with S.cerevisiae, P. stipitis, Z. mobilis, and C. thermocellum in a standardmicrobiological medium for each organism. YM broth is used for the twoyeast strains, S. cerevisiae and P. stipitis. RM medium is used for Z.mobilis and CM4 medium for C. thermocellum. A positive control, withpure sugar added, but no feedstock, is used for comparison. During theincubation, a total of five samples is taken over a 12 hour period attime 0, 3, 6, 9, and 12 hours and analyzed for viability (plate countsfor Z. mobilis and direct counts for S. cerevisiae) and ethanolconcentration.

Sugar content of the feedstocks is measured using High PerformanceLiquid Chromatography (HPLC) equipped with either a SHODEX® sugar SP0810or BIORAD AMINEX® HPX-87P column. Each of the feedstocks (approx. 5 g)is mixed with reverse osmosis (RO) water for 1 hour. The liquid portionof the mixture is removed and analyzed for glucose, galactose, xylose,mannose, arabinose, and cellobiose content. The analysis is performedaccording to National Bioenergy Center protocol Determination ofStructural Carbohydrates and Lignin in Biomass.

Phase 2: Cellulase Compatibility

Feedstocks are tested, in duplicate, with commercially availableACCELLERASE® 1000 enzyme complex, which contains a complex of enzymesthat reduces lignocellulosic biomass into fermentable sugars, at therecommended temperature and concentration in an Erlenmeyer flask. Theflasks are incubated with moderate shaking at around 200 rpm for 12hours. During that time, samples are taken every three hours at time 0,3, 6, 9, and 12 hours to determine the concentration of reducing sugars(Hope and Dean, Biotech J., 1974, 144:403) in the liquid portion of theflasks.

Example 26 Sugar Concentration Analysis Using HPLC

13 samples were analyzed for sugar concentration (HPLC) and toxicityagainst 3 microorganisms (Pichia stipitis, Saccharomyces cerevisiae, andZymomonas mobilis. Table 26 lists the equipment used for theseexperiments. Table 27 and 28 provide a list of the sugars (includingvendor and lot numbers) used to prepare the HPLC standard and theprotocol used to prepare the HPLC standard, respectively.

TABLE 26 Equipment Utilized in Experiments Equipment Manufacturer, NamepH meter Orion Shakers (2) B. Braun Biotech, Certomat BS-1 HPLC Waters,2690 HPLC Module Spectrophotometer Unicam, UV300 YSI Biochem AnalyzerInterscience, YSI

TABLE 27 Sugars used in HPLC analysis Sugar Manufacturer Ref # Lot #glucose BioChemika 49140 1284892 xylose 95731 1304473 51707231cellobiose 22150 1303157 14806191 arabinose 10840 1188979 24105272mannose 63582 363063/1 22097 galactose 48259 46032/1 33197

TABLE 28 Preparation of HPLC standards Desired Volume of TotalConcentration Volume of sugar Nanopure Water Volume (mg/mL) solution(mL) (mL) 4 50 mL of 4 mg/mL 0 50 2 25 mL of 4 mg/mL 25 50 1 25 mL of 2mg/mL 25 50 0.5 25 mL of 1 mg/mL 25 50 0.1 5 ml of 1 mg/mL 20 25Verification 18.75 mL of 4 mg/mL 31.25 50 Standard 1.5 mg/mL

Analysis

Each sample (1 gram) was mixed with reverse osmosis water at 200 rpm and50° C. overnight. The pH of the sample was adjusted to between 5 and 6and filtered through a 0.2 μm syringe filter. Samples were stored at−20° C. prior to analysis to maintain integrity of the samples. Theobservations made during the preparation of the samples are presented inTable 29.

TABLE 29 Observations During HPLC Sample Preparation Amount Water usedadded Sample (g) (mL) pH Observations P132 1 30 5.38 Fluffy, difficultto mix P132-10 1 25 6.77 Fluffy, difficult to mix P132-100 1 20 3.19 pHis low, difficult to bring to pH 5.0, used 10N NaOH P132-US 0.3 5 6.14A132 1 15 5.52 A132-10 1 15 4.9 A132-100 1 15 5.39 SG132 1 15 5.59SG132-10 1 15 5.16 SG132-100 1 15 4.7 SG132-10-US 0.3 5 5.12SG132-100-US 0.3 5 4.97 WS132 1 15 5.63 WS132-10 1 15 5.43 WS132-100 115 5.02 *pH of these samples was adjusted to pH using 1N NaOH

Standards were prepared fresh from a 4 mg/mL stock solution of the 6combined sugars, glucose, xylose, cellobiose, arabinose, mannose, andgalactose. The stock solution was prepared by dissolving 0.400 grams ofeach sugar into 75 mL of nanopure water (0.3 micron filtered). Oncedissolved, the stock solution was diluted to 100 mL using a volumetricflask and stored at −20° C. Working standard solutions of 0.1, 0.5, 1,2, and 4 mg/mL were prepared by serial dilution of the stock solutionwith nanopure water. In addition, a verification standard of 1.5 mg/mLwas also prepared from the stock solution.

Sugar concentrations were analyzed according to the protocolDetermination of Structural Carbohydrates in Biomass (NREL BiomassProgram, 2006) and this protocol is incorporated herein by reference inits entirety. A SHODEX SUGAR SP0810 COLUMN with an Evaporative LightScattering Detector was used. A verification standard (1.5 mg/mL ofstandard) was analyzed every 8 injections to ensure that the integrityof the column and detector were maintained during the experiment. Thestandard curve coefficient of variation (R² value) was at least 0.989and the concentration of the verification standards were within 10% ofthe actual concentration. The HPLC conditions were as follows:

TABLE 30 HPLC Parameters Injection volume: 20 μL Mobile phase: nanopurewater*, 0.45 μm filtered and degassed Flow rate: 0.5 mL/min Columntemperature: 85° C. Detector temperature: evaporator temperature 110°C., nebulizer temperature 90° C. *Initial tests noted that betterseparation was observed when using nanopure water than 15/85acetonitrile:water in the mobile phase (manufacturer does not recommendusing greater than 20% acetonitrile with this column).

Results

The results of the HPLC analysis are presented in Tables 31, 32, and 33.

TABLE 31 Sugar Concentration Expressed as mg/mL and mg/g of ExtractXylose Arabinose Glucose Galactose Mannose Cellobiose mW~150 mW~150mW~180 (see gluc) (see gluc) mW~342 C₅H₁₀O₅ C₅H₁₀O₅ C₆H₁₂O₆ mg/mL:mg/mL: C₁₂H₂₂O₁₁ Mono Mono Mono mg/g mg/g Disacc mg/ mg/ mg/ mg/ mg/ mg/Sample ID mL mg/g mL mg/g mL mg/g mL mg/g mL mg/g mL mg/g P P-132 0.000.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 P-132-10 0.000.00 0.00 0.00 0.34 8.60 0.00 0.00 0.00 0.00 00.33 8.13 P-132-100 0.357.04 0.00 0.00 0.34 6.14 0.00 0.00 0.00 0.00 0.36 7.20 P-132-BR 0.355.80 0.43 7.17 0.34 5.62 0.00 0.00 0.00 0.00 0.00 0.00 G G-132 0.39 5.880.38 5.73 0.84 12.66 0.34 5.04 0.92 13.76 0.00 0.00 G-132-10 0.50 7.500.41 6.18 1.07 16.04 0.35 5.19 0.98 14.66 0.00 0.00 G-132-100 0.00 0.000.37 5.54 0.41 6.14 0.00 0.00 0.55 8.28 0.45 6.71 G-132-10-US 0.34 5.730.39 6.45 0.33 5.43 0.00 0.00 0.00 0.00 0.00 0.00 G-132-100-US 0.00 0.000.37 6.22 0.35 5.90 0.33 5.43 0.40 6.70 0.39 6.45 A A-132 1.36 20.390.00 0.00 1.08 16.22 0.39 5.84 1.07 16.02 0.00 0.00 A-132-10 1.19 17.870.00 0.00 0.00 0.00 0.00 0.00 0.37 5.52 0.00 0.00 A132-100 1.07 16.110.00 0.00 0.35 5.18 0.00 0.00 0.00 0.00 0.81 12.2 WS WS-132 0.49 7.410.41 6.15 0.39 5.90 0.00 0.00 0.00 0.00 0.00 0.00 WS-132-10 0.57 8.490.40 5.99 0.73 10.95 0.34 5.07 0.50 7.55 0.00 0.00 WS-132-100 0.43 6.390.37 5.51 0.36 5.36 0.00 0.00 0.36 5.33 0.35 5.25

TABLE 32 Sugar Concentration Expressed at % of Paper Sugar concentra-tion (% of dry sample) P132 P132-10 P132-100 P132-US cellobiose 0.000.81 0.72 0.00 glucose 0.00 0.86 0.67 0.56 xylose 0.00 0.00 0.70 0.58galactose 0.00 0.00 0.00 0.00 arabinose 0.00 0.00 0.00 0.72 mannose 0.000.00 0.00 0.00

TABLE 33 Sugar Concentration Expressed at % of Total Sample Sugarconcentration (% of dry A132- A132- A132- SG132- SG132- SG132- SG132-WS132- WS132- sample) A132 10 100 SG132 10 100 10-US 100-US WS132 10 100cellobiose 0.00 0.00 1.22 0.00 0.00 0.67 0.00 0.65 0.00 0.00 0.53glucose 1.62 0.00 0.52 1.27 1.60 0.61 0.54 0.59 0.59 1.10 0.54 xylose2.04 1.79 1.61 0.59 0.75 0.00 0.57 0.00 0.74 0.85 0.64 galactose 0.580.00 0.00 0.50 0.52 0.00 0.00 0.54 0.00 0.51 0.00 arabinose 0.00 0.000.00 0.57 0.62 0.55 0.65 0.62 0.62 0.60 0.55 mannose 1.60 0.55 0.00 1.381.47 0.83 0.00 0.67 0.00 0.76 0.53

Example 27 Toxicity Study

Twelve samples were analyzed for toxicity against a panel of threeethanol-producing cultures. In this study, glucose was added to thesamples in order to distinguish between starvation of the cultures andtoxicity of the samples. A thirteenth sample was tested for toxicityagainst Pichia stipitis. A summary of the protocol used is listed inTable 32. A description of the chemicals and equipment used in thetoxicity testing is reported in Tables 34-36.

TABLE 34 Conditions for Toxicity Testing Organism ZymomonasSaccharomyces Pichia mobilis cerevesiae stipitis ATCC ATCC NRRL Variable31821 24858 Y-7124 Test Repetition Duplicate Inoculation  1   0.1  1Volume (mL) Incubation 30° C. 25° C. 25° C. Temperature Shaker Speed(rpm) 125 200  125 Erlenmeyer 250 mL 500 mL 250 mL Flask Volume Mediavolume 100 mL 100 mL 100 mL Total Incubation  36 36  48 time (hours)Ethanol Analysis 24, 30, 36 24, 30, 36 24, 36, 48 (hours) Cell Counts24, 36 24, 36 24, 48 (hours) pH 0 hours 0 hours 0 hours

TABLE 35 Reagents Used for Toxicity Testing Media Component ManufacturerReference # Lot # Urea ScholAR Chemistry 9472706 AD-7284-43 YeastNitrogen Base Becton Dickinson 291940 7128171 Peptone Becton Dickinson211677 4303198 Xylose Fluka 95731 1304473 51707231 Glucose Sigma G-5400107H0245 Yeast Extract Becton Dickinson 288620 4026828 (used for S.cerevisiae) Yeast Extract (used Becton Dickinson 212750 7165593 for P.stipitis and Z. mobilis) MgSO₄•7H₂O Sigma M5921 034K0066 (NH₄)₂SO₄ SigmaA4418 117K5421 KH₂PO₄ Sigma P5379 074K0160 YM Broth Becton Dickinson271120 6278265

TABLE 36 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 g/L)2790 012711040 YSI Ethanol Buffer 2787 07M1000053, 07100215

Testing was performed using the three microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A slant of S. cerevisiae was prepared from a rehydrated lyophilizedculture obtained from ATCC. A portion of the slant material was streakedonto an YM Broth+20 g/L agar (pH 5.0) and incubated at 30° C. for 2days. A 250 mL Erlenmeyer flask containing 50 mL of medium (20 g/Lglucose, 3 g/L yeast extract, and 5.0 g/L peptone, pH 5.0) wasinoculated with one colony from the YM plate and incubated for 24 hoursat 25° C. and 200 rpm. After 23 hours of growth, a sample was taken andanalyzed for optical density (600 nm in a UV spectrophotometer) andpurity (Gram stain). Based on these results, two seed flasks, eachhaving an optical density (OD) of between 4 and 8 and with a clean Gramstain, were combined to inoculate the growth flasks.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving will change the content ofthe samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. The flasks were incubated as described above for 36hours.

Pichia stipitis NRRL Y-7124 (ARS Culture Collection) A slant of P.stipitis was prepared from a rehydrated lyophilized culture obtainedfrom ARS Culture Collection. A portion of the slant material wasstreaked onto an YM Broth+20 g/L agar (pH 5.0) and incubated at 30° C.for 2 days. A 250 mL Erlenmeyer flask containing 100 mL of medium (40g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/Lpeptone, 40 g/L xylose, pH 5.0) was inoculated with a small amount ofplate material and incubated for 24 hours at 25° C. and 125 rpm. After23 hours of growth, a sample was taken and analyzed for optical density(600 nm in a UV spectrophotometer) and purity (Gram stain). Based onthese results, one flask (called the Seed Flask) at an optical densityof 5.23 and with a clean Gram Stain was chosen to inoculate all of thetest flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22 μm filter) media added to theflasks prior to the addition of the test materials. The test materialswere not sterilized, as autoclaving will change the content of thesamples and filter sterilization not appropriate for sterilization ofsolids. The test samples were added at the time of inoculation (ratherthan prior to) to reduce the possibility of contamination. In additionto the test samples, 1 mL (1% v/v) of seed flask material was added toeach flask. The flasks were incubated as described above for 48 hours.

Zymomonas mobilis ATCC 31821 (American Type Culture)

A slant of Z. mobilis was prepared from a rehydrated lyophilized cultureobtained from ATTC. A portion of the slant material was streaked onto anDYE plates (glucose 20 g/L, Yeast Extract 10 g/L, Agar 20 g/L, pH 5.4)and incubated at 30° C. and 5% CO₂ for 2 days. A 20 mL screw-cap testtube containing 15 mL of medium (25 g/L glucose, 10 g/L yeast extract, 1g/L MgSO₄.7H₂O, 1 g/L (NH₄)₂SO₄, 2 g/L KH₂PO₄, pH 5.4) was inoculatedwith one colony and incubated for 24 hours at 30° C. with no shakingAfter 23 hours of growth, a sample was taken and analyzed for opticaldensity (600 nm in a UV spectrophotometer) and purity (gram stain).Based on these results, one tube (OD 1.96) was chosen to inoculate thesecond seed flask. The second seed flask was a 125 ml flask containing70 mL of the media described above and was inoculated with 700 μL (1%v/v) and incubated for 24 hours at 30° C. with no shaking After 23 hoursof growth, a sample was taken and analyzed for optical density (600 nmin a UV spectrophotometer) and purity (gram stain). Based on theseresults, one flask (called the Seed Flask) with an OD of 3.72 was chosento inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above with the exception of yeast extract at 5g/L. All flasks were autoclaved empty at 121° C. and 15 psi and filtersterilized (0.22 μm filter) media added to the flasks prior to theaddition of the test materials. The test materials were not sterilized,as autoclaving will change the content of the samples and filtersterilization not appropriate for sterilization of solids. The testsamples were added at the time of inoculation to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated asdescribed above for 36 hours

Analysis

Two samples were analyzed for cell concentration (using spread platingfor Z. mobilis and direct counts (haemocytometer and microscope for S.cerevisiae and P. stipitis). Appropriately diluted samples of Z. mobiliswere spread on Dextrose Yeast Extract (glucose 20 g/L, Yeast Extract 10g/L, Agar 20 g/L, pH 5.4) plates, incubated at 30° C. and 5% CO2 for 2days, and the number of colonies counted. Appropriately diluted samplesof S. cerevisiae and P. stipitis were mixed with 0.05% Trypan blue,loaded into a Neubauer haemocytometer. The cells were counted under 40×magnification.

Three samples were analyzed for ethanol concentration using the YSIBiochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. to preserve integrity. The sampleswere diluted to between 0-3.2 g/L ethanol prior to analysis. A standardof 3.2 g/L ethanol was analyzed approximately every 30 samples to ensurethe integrity of the membrane was maintained during analysis. Theoptical density (600 nm) of the samples is not reported because thesolid test samples interfered with absorbance measurement by increasingthe turbidity of the samples and are inaccurate.

Results of Ethanol Analysis

Performance was used to compare each sample to the control for eachmicroorganism (Tables 37-39). However, the % performance cannot be usedto compare between strains. When comparing strains, the totalconcentration of ethanol should be used. When analyzing the data, a %performance of less than 80% may indicate toxicity when accompanied bylow cell number. The equation used to determine % performance is:

% Performance=(ethanol in the sample/ethanol in control)×100

TABLE 37 Ethanol Concentration and % Performance Using Saccharomycescerevisiae 24 hours 30 hours 36 hours Ethanol Ethanol Ethanol Concen- %Concen- % Concen- % Sample tration Perform- tration Perform- trationPerform- # (g/L) ance (g/L) ance (g/L) ance P132 4.0 140 5.2 127 3.26176 P132- 4.2 147 5.1 125 3.86 209 10 P132- 4.3 149 5.6 136 3.47 187 100A132 5.5 191 6.5 160 5.24 283 A132- 1.9 67 6.3 153 5.54 299 10 A132- 4.4154 5.6 137 4.04 218 100 G132 5.3 186 6.0 146 3.99 215 G132- 5.2 180 6.4156 4.63 250 10 G132- 5.5 191 6.3 155 4.60 248 100 WS132 4.8 168 6.3 1554.51 244 WS132- 4.9 172 6.0 146 4.55 246 10 WS132- 4.9 170 5.7 140 4.71254 100 Control 2.9 100 4.1 100 1.85 100

TABLE 38 Ethanol Concentration and % Performance Using Pichia stipitis24 hours 36 hours 48 hours Ethanol Ethanol Ethanol Concen- % Concen- %Concen- % Sample tration Perform- tration Perform- tration Perform- #(g/L) ance (g/L) ance (g/L) ance P132 2.8 130 3.4 188 8.1 176 P132- 7.3344 11.9 655 15.8 342 10 P132- 5.2 247 8.6 472 13.3 288 100 A132 12.2575 14.7 812 14.9 324 A132- 15.1 710 18.7 1033 26.0 565 10 A132- 10.9514 16.7 923 22.2 483 100 G132 8.0 375 12.9 713 13.3 288 G132- 10.1 47616.0 884 22.3 485 10 G132- 8.6 406 15.2 837 21.6 470 100 WS132 9.8 46014.9 820 17.9 389 WS132- 7.8 370 16.1 890 19.3 418 10 WS132- 9.1 42915.0 829 15.1 328 100 Sample 13.2 156 19.0 166 20.6 160 A* Control 2.1100 1.8 100 4.6 100 Samples in BOLD were the highest ethanol producers,over 20 g/L and similar to the concentrations in wood hydrolyzates (H.K.Sreenath and T.W. Jeffries Bioresource Technology 72 (2000) 253-260).*Analyzed in later shake flask experiment.

TABLE 39 Ethanol Concentration and % Performance Using Zymomonas mobilis24 hours 30 hours 36 hours Ethanol Ethanol Ethanol Concen- % Concen- %Concen- % Sample tration Perform- tration Perform- tration Perform- #(g/L) ance (g/L) ance (g/L) ance P132 7.5 85 6.8 84 7.5 93 P132- 7.5 854.8 59 6.8 84 10 P132- 7.3 83 6.2 77 7.1 88 100 A132 9.6 109 8.3 103 9.1112 A132- 9.2 105 8.4 105 8.8 109 10 A132- 8.2 93 7.6 94 7.6 93 100WS132 7.9 89 7.1 88 7.7 94 WS132- 8.2 93 6.8 85 7.3 90 10 WS132- 8.7 986.9 86 8.3 102 100 G132 8.7 99 7.1 88 8.1 99 G132- 7.8 88 7.0 88 7.3 9010 G132- 8.6 98 7.8 98 8.3 102 100 Control 8.8 100 8.0 100 8.1 100Results from Cell Concentration Analysis

% Cells is used to compare each sample to the control for each organism(Tables 40-42). However, the % cells cannot be used to compare betweenstrains. When comparing strains, the total concentration of cells shouldbe used. When analyzing the data, a % performance of less than 70% mayindicate toxicity when accompanied by low ethanol concentration. Theequation used to determine % performance is:

% cells=(number of cell in the sample/number of cells in control)×100

TABLE 40 Results from Cell Concentration Analysis for Saccharomycescerevisiae 24 hours 36 hours Cell Concentration % Cell Concentration %Sample # (×10⁸/mL) Cells (×10⁸/mL) Cells P132 1.99 166 2.51 83 P132-102.51 209 1.91 63 P132-100 1.35 113 1.99 66 A132 3.80 316 2.59 85 A132-101.73 144 3.90 129 A132-100 3.98 331 2.51 83 G132 2.14 178 3.12 103G132-10 2.33 194 2.59 85 G132-100 3.57 298 2.66 88 WS132 4.10 341 2.6688 WS132-10 2.63 219 2.81 93 WS132-100 2.29 191 2.40 79 Control 1.20 1003.03 100

TABLE 41 Results from Cell Concentration Analysis for Pichia stipitis 24hours 48 hours Cell Concentration % Cell Concentration % Sample #(×10⁸/mL) Cells (×10⁸/mL) Cells P132 16.4 108 20.3 87 P132-10 11.5 769.5 41 P132-100 6.5 43 17.8 76 A132 7.1 47 10.2 44 A132-10 12.7 84 9.340 A132-100 11.8 78 18.3 78 G132 4.5 30 4.8 21 G132-10 22.8 151 9.8 42G132-100 10.1 67 21.7 93 WS132 17.6 117 8.2 35 WS132-10 5.3 35 10.8 46WS132-100 9.3 62 10.7 46 Control 15.1 100 23.4 100

TABLE 42 Results from Cell Concentration Analysis for Zymomonas mobilis24 hours 36 hours Cell Concentration % Cell Concentration % Sample #(×10⁸/mL) Cells (×10⁸/mL) Cells P132 7.08 86 2.97 66 P132-10 21.80 2644.37 98 P132-100 4.50 54 3.35 75 A132 6.95 84 1.99 44 A132-10 6.13 744.05 91 A132-100 9.60 116 4.20 94 G132 7.48 90 3.84 86 G132-10 14.75 1782.89 65 G132-100 6.00 72 2.55 57 WS132 9.70 117 4.55 102 WS132-10 13.20160 4.32 97 WS132-100 5.15 62 2.89 65 Control 8.27 100 4.47 100

Example 28 Shake Flask Fermentation of Cellulose Samples Using P.stipitis Summary

Thirteen samples were tested for ethanol production in P. stipitisculture without sugar added. They were tested in the presence andabsence of cellulase (ACCELLERASE® 1000, Genencor). Equipment andreagents used for the experiment are listed below in Tables 43-45.

TABLE 43 Equipment and frequency of maintenance Frequency of EquipmentManufacturer Maintenance Shakers (2) B. Braun Biotech, QuarterlyCertomat BS-1 Spectrophotometer Unicam, UV300 Biannual YSI BiochemAnalyzer Interscience, YSI Monthly

TABLE 44 YSI Components used in shake flask study Component Catalog #Lot # YSI Ethanol Membrane 2786 07L100153 YSI Ethanol Standard (3.2 g/L)2790 012711040 YSI Ethanol Buffer 2787 07M1000053, 07100215

TABLE 45 Chemicals used for shake flask fermentation Media ComponentManufacturer Reference # Lot # Urea ScholAR 9472706 AD-7284-43 ChemistryYeast Nitrogen Becton 291940 7128171 Base Dickinson Peptone Becton211677 4303198 Dickinson YM Broth Becton 271120 6278265 DickinsonACCELLERASE ® Genencor ACCELLERASE ® 1600794133 Enzyme complex 1000Xylose BioChemika 95731 1304473 51707231 Glucose Sigma G-5400 107H0245

A slant of P. stipitis NRRL Y-7124 was prepared from a rehydratedlyophilized culture obtained from ARS Culture Collection. A portion ofthe slant material was streaked onto a Yeast Mold (YM) Broth+20 g/L agar(pH 5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 100 mL of medium (40 g/L glucose, 1.7 g/L yeast nitrogenbase, 2.27 g/L urea, 6.56 g/L peptone, 40 g/L xylose, pH 5.0) wasinoculated with one colony and incubated for 24 hours at 25° C. and 100rpm. After 23 hours of growth, a sample was taken and analyzed foroptical density (600 nm in a UV spectrophotometer) and purity (Gramstain). Based on these results, one flask (called the Seed Flask) at anoptical density of 6.79 and with a clean Gram stain was chosen toinoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL ofmedium (1.7 g/L yeast nitrogen base, 2.27 g/L urea, and 6.56 g/Lpeptone). No sugar (glucose or xylose) was added to the growth flaskmedium. All flasks were autoclaved empty at 121° C. and 15 psi andfilter sterilized (0.22 μm filter) media added to the flasks prior tothe addition of the test materials. The test materials were notsterilized, as autoclaving will change the content of the samples andfilter sterilization is not appropriate for sterilization of solids. Thetest samples (listed in Table 46) were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 1 mL (1% v/v) of seed flask material wasadded to each flask. Flasks containing sample P132-100 required theaddition of 0.4 mL 1 M NaOH to bring the pH to 5.0. The flasks wereincubated at 30° C. and 150 rpm above for 96 hours.

One set of duplicate flasks per feedstock contained ACCELLERASE® enzymecomplex (1.25 mL per flask, highest recommended dosage is 0.25 mL pergram of biomass, Genencor) to attempt simultaneous saccharification andfermentation (SSF). The other set of duplicate flasks did not containACCELLERASE® enzyme complex. A total of 52 flasks were analyzed.

Six control flasks were also analyzed. Positive control flasks containedSolkaFloc 200 NF Powdered Cellulose (lot # UA158072, International FiberCorporation) at a concentration of 2.5 grams per 100 mL flask (25 gramsper L) with and without addition of ACCELLERASE® enzyme complex. Inaddition, a control containing sugars (glucose and xylose) only wasused.

TABLE 46 The amount of each feedstock added to each flask Amount addedto Flask Xyleco Number (g/100 mL) P132 2.5 P132-10 2.5 P132-100 2.5 A1325 A132-10 5 A132-100 5 G132 5 G132-10 5 G132-100 5 WS132 5 WS132-10 5WS132-100 5 Sample A 5

Analysis

Samples were analyzed for ethanol concentration (Tables 47, 48, and 49)using the YSI Biochem Analyzer based on the alcohol dehydrogenase assay(YSI, Interscience). Samples were centrifuged at 14,000 rpm for 20minutes and the supernatant stored at −20° C. The samples were dilutedto between 0-3.2 g/L ethanol prior to analysis. A standard of 2.0 g/Lethanol was analyzed approximately every 30 samples to ensure theintegrity of the membrane was maintained during analysis.

Results

TABLE 47 Results of Control Flasks Ethanol Concentration (g/L) Control24 hours 36 hours 48 hours 96 hours Containing Glucose, no 13.20 19.0020.60 21.60 cellulose, no enzyme Containing Crystalline 0.00 0.00 0.000.00 Cellulose (Solka Floc), no sugar, no enzyme Containing Crystalline6.56 7.88 9.80 8.65 Cellulose (Solka Floc) at 25 g/L, no sugar,Accellerase ® added

TABLE 48 Results of Shake Flasks without ACCELLERASE ® 1000 EthanolConcentration (g/L) Sample Number 24 hours 36 hours 48 hours 96 hoursP132 0.09 0.00 0.00 0.12 P132-10 0.02 0.01 0.02 0.17 P132-100 0.09 0.010.00 0.02 A132 1.74 1.94 2.59 3.70 A132-10 1.82 2.36 2.30 2.96 A132-1000.30 0.73 1.31 2.38 G132 0.40 0.09 0.24 0.42 G132-10 0.69 0.42 0.22 0.24G132-100 0.19 0.05 0.05 0.21 WS132 0.47 0.50 0.68 0.65 WS132-10 0.470.49 0.34 0.92 WS132-100 0.14 0.07 0.08 0.22 Sample A 1.88 1.89 2.303.28

TABLE 49 Results of Shake Flasks with ACCELLERASE ® 1000 EthanolConcentration (g/L) Sample Number 24 hours 36 hours 48 hours 96 hoursP132 7.04 8.72 9.30 5.80 P132-10 4.22 4.48 4.49 1.24 P132-100 3.18 4.284.70 3.35 A132 2.79 2.91 2.03 4.30 A132-10 3.31 1.62 2.11 2.71 A132-1002.06 1.92 1.02 1.47 G132 0.87 0.40 0.32 0.44 G132-10 1.38 1.04 0.63 0.07G132-100 2.21 2.56 2.34 0.12 WS132 1.59 1.47 1.07 0.99 WS132-10 1.921.18 0.73 0.23 WS132-100 2.90 3.69 3.39 0.27 Sample A 2.21 2.35 3.392.98

Example 29 Cellulase Assay Summary

Thirteen samples were tested for cellulase susceptibility using anindustry cellulase (ACCELLERASE® 1000, Genencor) under optimumconditions of temperature and pH.

Protocol

The protocol is a modification of the NREL “Laboratory AnalyticalProcedure LAP-009 Enzymatic Saccharification of LignocellulosicBiomass”. A sample of material was added to 10 mL 0.1 M sodium citratebuffer (pH 4.8) and 40 mg/mL tetracycline (to prevent growth ofbacteria) in a 50 mL tube in duplicate. The amount of sample added toeach tube is listed in Table 50. Some samples were difficult to mix(P132, P132-10, P132-100), so were added at a lower concentration. Apositive control of 0.2 grams SolkaFloc 200 NF Powdered Cellulose (lot #UA158072, International Fiber Corporation) and a negative control (nosample) were also included. Enough reverse osmosis (RO) water to bringthe volume to a total of 20 mL was added to the tubes. Both the sodiumcitrate buffer and water were heated to 50° C. prior to use.

ACCELLERASE® 1000 enzyme was added to each tube at a dosage of 0.25 mLper gram of biomass (highest dosage recommended by Genecor). The tubeswere incubated at 45° angle at 150 rpm and 50 degrees C. (recommended byGenencor) for 72 hours. Samples were taken at 0, 3, 6, 9, 12, 18, 24,48, and 72 hours (Table 52 and 53), centrifuged at 14,000 rpm for 20minutes and the supernatant frozen at −20° C. The glucose concentrationin the samples was analyzed using the YSI Biochem Analyzer(Interscience) using the conditions described in Table 51. A glucosestandard solution of 2.5 g/L was prepared by dissolving 2.500 gramsglucose (Sigma Cat# G7528-5KG, Lot#:107H0245) in distilled water. Oncedissolved, the total volume was brought to 1 L with distilled water in avolumetric flask. The standard was prepared fresh weekly and stored at4° C.

TABLE 50 Amount of Each Sample Added Xyleco Number Amount added to Tube(g/20 mL) P132 0.5 P132-10 0.5 P132-100 0.5 A132 0.75 A132-10 0.75A132-100 0.75 G132 0.75 G132-10 0.75 G132-100 0.75 WS132 0.75 WS132-100.75 WS132-100 0.75 Sample A 0.75 SolkaFloc 200NF (Control) 0.2 NegativeControl 0

TABLE 51 YSI Components Used in Shake Flask Study Component Catalog #Lot # YSI Glucose Membrane 2365 07D100124 YSI Glucose Buffer 2357014614A

Results

TABLE 52 Cellulase Assay Results Sample Glucose Concentration (mg/mL) atIncubation Time (hours) Number 0 3 6 9 12 18 24 48 72 P132 0.59 4.197.00 8.72 9.70 10.95 12.19 15.10 15.65 P132- 0.36 3.37 5.08 6.39 6.987.51 8.99 11.25 11.65 10 P132- 0.91 3.86 5.67 7.31 8.08 9.47 10.70 12.7013.80 100 A132 0.39 1.51 1.92 2.40 2.64 3.04 3.30 3.90 4.06 A132- 0.421.80 2.27 2.63 2.86 3.16 3.43 4.02 4.14 10 A132- 0.46 2.09 2.72 3.163.43 3.78 4.09 4.84 5.26 100 G132 0.40 1.16 1.35 1.52 1.60 1.67 1.852.10 2.21 G132- 0.34 1.34 1.64 1.95 2.03 2.09 2.36 2.77 3.02 10 G132-0.61 1.84 2.32 2.89 3.14 3.52 3.97 4.81 5.44 100 WS132 0.35 1.48 1.812.14 2.26 2.50 2.70 3.18 3.26 WS132- 0.44 1.77 2.22 2.60 2.76 2.61 3.153.62 3.82 10 WS132- 0.70 2.76 3.63 4.59 4.78 5.29 5.96 6.99 7.43 100Sample A 0.42 1.09 1.34 1.55 1.69 1.66 2.17 2.96 3.71 Negative Control(no sample) 0.03 0.03 0.01 0.01 0.02 0.01 0.02 0.02 0.02 PositiveControl 0.17 2.38 3.65 4.71 5.25 5.98 7.19 9.26 9.86 (SolkaFloc)

FIG. 65 shows a graph of glucose concentration (top 4 producers).

The amount of cellulose digested in the tube was calculated as follows:

g/mL glucose×20 mL (volume of sample)×0.9 (to correct for the watermolecule added upon hydrolysis of cellulose)

The percent of the total sample released as glucose (in Table 53 below)was calculated as follows:

g of cellulose digested/g of sample added (see Table 5 for details)*100

TABLE 53 Cellulase Assay Results Percent of the Total Sample Released asSample Glucose (%) at Incubation Time (h) Number 0 3 6 9 12 18 24 48 72P132 2.02 14.98 25.16 31.36 34.85 39.38 43.81 54.29 56.27 P132- 1.1912.02 18.25 22.97 25.06 27.00 32.29 40.43 41.87 10 P132- 3.17 13.7920.38 26.28 29.02 34.06 38.45 45.65 49.61 100 A132 0.86 3.55 4.58 5.746.29 7.27 7.87 9.31 9.70 A132- 0.94 4.25 5.42 6.29 6.82 7.56 8.18 9.609.89 10 A132- 1.03 4.94 6.50 7.56 8.18 9.05 9.77 11.57 12.58 100 G1320.89 2.71 3.22 3.62 3.79 3.98 4.39 4.99 5.26 G132- 0.74 3.14 3.91 4.664.82 4.99 5.62 6.60 7.20 10 G132- 1.39 4.34 5.54 6.91 7.49 8.42 9.4811.50 13.01 100 WS132 0.77 3.48 4.32 5.11 5.38 5.98 6.43 7.58 7.78WS132- 0.98 4.18 5.30 6.22 6.58 6.24 7.51 8.64 9.12 10 WS132- 1.61 6.558.69 10.99 11.42 12.67 14.26 16.73 17.78 100 Sample A 0.94 2.54 3.193.70 4.01 3.96 5.16 7.06 8.86 Positive Control 1.29 21.15 32.72 42.3047.07 53.73 64.53 83.16 88.56 (Solka Floc)

Example 30 Shake Flask Fermentation Using Pichia stipitis Summary

Shake flask fermentation using Pichia stipitis was performed using fourcellulosic materials having the highest % performance from Table 36.

Protocol

Experiments were run under the parameters outlined in Tables 54-56.

TABLE 54 Equipment and Frequency of Maintenance Frequency of EquipmentManufacturer, Name Maintenance Shakers (2) B. Braun Biotech, QuarterlyCertomat BS-1 Spectrophotometer Unicam, UV300 Biannual YSI BiochemAnalyzer Interscience, YSI Monthly

TABLE 55 YSI Components Used in Shake Flask Study Component Reference #Lot # YSI Ethanol Membrane 2786 07M100361 YSI Ethanol Standard (3.2 g/L)2790 1271040 YSI Ethanol Buffer 2787 07J100215

TABLE 56 Chemicals Used for Shake Flask Fermentation Media ComponentManufacturer Reference # Lot # Urea ScholAR 9472706 AD-7284-43 ChemistryYeast Nitrogen Becton Dickinson 291940 7128171 Base Peptone BectonDickinson 211677 4303198 YM Broth Becton Dickinson 271120 6278265 XyloseAlfa Aesar A10643 10130919 Glucose Fisher Scientific BP350-1 030064

Seed Development

For all the following shake flask experiments the seed flasks wereprepared using the following procedure.

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol were storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for 2 days at 4° C. beforeuse. A 250 mL Erlenmeyer flask containing 100 mL of medium (40 g/Lglucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone,40 g/L xylose, pH 5.0) was inoculated with one colony and incubated for24 hours at 25° C. and 100 rpm. After 23 hours of growth, a sample wastaken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of between 4 and 8and with a clean Gram stain was used to inoculate all of the testflasks.

Three experiments were run using samples A132-10, A132-100, G132-10, andG132-100. Experiment #1 tested these four samples for ethanolconcentration at varying concentrations of xylose and at constantconcentrations of glucose. Experiment #2 tested these four samples forethanol concentration at double the concentration of feedstock used inthe experiments of Table 36. Finally, experiment #3 tested these foursamples for ethanol concentration while varying both the xylose and theglucose concentrations, simultaneously.

Experiment #1 Varying the Xylose Concentration

Four cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose concentrations as listed in Table 57 below.

TABLE 57 Media Composition of Experiment #1 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 100% Xylose 40.0 40.0 50%Xylose 20.0 40.0 25% Xylose 10.0 40.0 10% Xylose 4.0 40.0  0% Xylose 0.040.0

The test vessels (a total of 40, 250 mL Erlenmeyer flasks) contained 100mL of medium. Five different types of media were prepared with theamount of xylose and glucose outlined in Table 57. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson #291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). All flasks were autoclaved empty at 121° C. and 15psi and filter sterilized (0.22 μm filter) media was added to the flasksprior to the addition of the test materials. Flasks were held at roomtemperature for 4 days and inspected for contamination (cloudiness)prior to use. The test materials were not sterilized, as autoclavingwill change the content of the samples and filter sterilization notappropriate for sterilization of solids. The test samples (A132-10,A132-100, G132-10, and G132-100 at 5 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Unfortunately, one flask (sample A132-100 with 100% Xylose) was brokenduring the testing. Therefore, all results past 24 hours of incubationare reported as a single flask. After 72 hours of incubation, 100% ofthe original amount of cellulosic material (5.0 g) was added to the 100%Xylose flasks (7 flasks in total, one flask containing sample A132-100was broken) and incubated as above for an additional 48 hours.

TABLE 58 Addition of Feedstock to 100% Xylose Flasks at Incubation Time72 hours Feedstock Added at 72 hours (grams) A132-10 5 A132-100 5G132-10 5 G132-100 5

Analysis

Samples were taken from the 40 test flasks at incubation times of 0, 6,12, 24, 36, 48, and 72 hours. In addition, samples were taken at 24 and48 hours post-addition of the second feedstock amount in the 100% Xyloseflasks (see Table 58).

A total of 292 samples were analyzed for ethanol concentration using aYSI Biochem Analyzer based on the alcohol dehydrogenase assay (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. Of note, time 0 samples requiredfiltration through a 0.45 μm syringe filter. The samples will be dilutedto between 0-3.2 g/L ethanol prior to analysis. A standard of 2.0 g/Lethanol was analyzed approximately every 30 samples to ensure theintegrity of the membrane was maintained.

A total of 47 samples were analyzed for cell count. Samples will betaken at 72 hours incubation and 48 hours post-addition of morecellulosic material. Appropriately diluted samples were mixed with 0.05%Trypan blue and loaded into a Neubauer haemocytometer. The cells werecounted under 40× magnification.

Experiment #2 Analysis of 2× Feedstock Concentration

The test vessels (a total of 8, 250 mL Erlenmeyer flasks) contained 100mL of medium. The media contained 40 g/L glucose, 40 g/L xylose, 1.7 g/Lyeast nitrogen base (Becton Dickinson #291940) 2.27 g/L urea (ScholARChemistry #9472706), and 6.56 g/L peptone (Becton Dickinson #211677).Flasks were prepared as in Experiment #1. The test samples (A132-10,A132-100, G132-10, and G132-100 at 10 g per 100 mL) were added at thetime of inoculation (rather than prior to) to reduce the possibility ofcontamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm above for 72 hours.

Analysis

Samples were from the 8 test flasks at an incubation time of 0, 6, 12,24, 36, 48, and 72 hours. Ethanol analyses of the 56 samples wasperformed as per experiment #1 and are reported in Table 59. A cellcount was performed on the 72 hour sample as per experiment #1 and ispresented in Table 60.

TABLE 59 Ethanol Concentration in Flasks with Double Feedstock EthanolConcentration (g/L) Sample Time A132-10 A132-100 G132-10 G132-100 0 1.380.26 0.12 0.11 6 1.75 0.21 0.20 0.10 12 2.16 0.73 0.69 0.31 24 19.0515.35 16.55 12.60 36 21.75 17.55 18.00 15.30 48 26.35 23.95 24.65 20.6572 26.95 27.35 28.90 27.40

TABLE 60 Cell Concentration at 72 hour Incubation Time in Flasks withDouble Feedstock Sample Cell Concentration (×10⁸/mL) A132-10 4.06A132-100 5.37 G132-10 5.18 G132-100 4.47

Experiment #3 Varying Xylose and Glucose Concentrations

Four cellulosic samples (A132-10, A132-100, G132-10, and G132-100) weretested at varying xylose and glucose concentrations as listed in thetable below (Table 60).

TABLE 61 Media Composition of Experiment #3 Flasks Xylose ConcentrationGlucose Concentration Treatment (g/L) (g/L) 50% Sugar 20.0 20.0 25%Sugar 10.0 10.0 10% Sugar 4.0 4.0  0% Sugar 0.0 0

The test vessels (a total of 32, 250 mL Erlenmeyer flasks) contained 100mL of medium. Four different types of media were prepared with theamount of xylose and glucose outlined in Table 61. In addition, themedia contained 1.7 g/L yeast nitrogen base (Becton Dickinson #291940)2.27 g/L urea (ScholAR Chemistry #9472706), and 6.56 g/L peptone (BectonDickinson #211677). The flasks were prepared as per Experiment #1. Thetest samples (A132-10, A132-100, G132-10, and G132-100) were added atthe time of inoculation (rather than prior to) to reduce the possibilityof contamination. In addition to the test samples, 1 mL (1% v/v) of seedflask material was added to each flask. The flasks were incubated at 30°C. and 150 rpm for 72 hours.

Analysis

Samples were taken from the 32 test flasks at an incubation time of 0,6, 12, 24, 36, 48, and 72 hours (see Tables 62-65). A total of 224samples were analyzed for ethanol concentration using the YSI BiochemAnalyzer based on the alcohol dehydrogenase assay (YSI, Interscience).Samples were centrifuged at 14,000 rpm for 20 minutes and thesupernatant stored at −20° C. Of note, some of the samples requiredcentrifugation and then filtration through a 0.45 μm syringe filter. Thesamples were diluted to between 0-3.2 g/L ethanol prior to analysis. Astandard of 2.0 g/L ethanol was analyzed approximately every 30 samplesto ensure the integrity of the YSI membrane was maintained.

TABLE 62 Ethanol Results Sample A132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.43 0.42 0.42 0.41 0.390.53 0.57 0.56 0.56 6 1.16 1.16 1.15 1.16 1.12 0.93 0.91 0.83 0.88 121.72 1.86 1.71 1.79 1.90 1.21 2.13 2.47 2.32 24 15.55 15.90 17.05 17.0516.95 1.02 4.88 9.77 13.35 36 17.10 17.40 20.25 21.35 20.25 1.29 4.279.99 17.55 48 16.40 17.05 19.70 23.00 26.80 1.47 3.03 8.33 16.60 7215.15 15.55 19.25 21.85 28.00 1.14 1.52 5.08 14.20 24 — — — — 23.15 — —— — hours post- addition 48 — — — — 21.55 — — — — hours post- addition*Analysis from experiment #3.

TABLE 63 Ethanol Results Sample A132-100 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.11 0.09 0.17 0.20 0.180.12 0.14 0.09 0.13 6 0.13 0.15 0.15 0.15 0.14 0.10 0.11 0.11 0.13 120.88 1.00 1.18 1.25 0.89 0.18 1.58 1.55 1.57 24 15.90 15.70 16.50 16.0514.60** 0.18 3.33 7.99 11.15 36 16.00 17.90 16.90 19.45 17.80** 0.212.85 8.37 16.10 48 15.75 16.70 19.30 22.15 27.00** 0.54 1.47 7.54 15.6072 14.85 15.35 18.55 21.30 28.50** 0.78 0.51 4.47 12.90 24 hours — — —24.80** — — — — — post- addition 48 hours — — — 23.60** — — — — — post-addition *Analysis from experiment #3. **All results based on analysisof one flask.

TABLE 64 Ethanol Results Sample G132-10 Ethanol Concentration (g/L)Sample 0% 10% 25% 50% 100% 0% 10% 25% 50% Time Xylose Xylose XyloseXylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.09 0.08 0.08 0.08 0.080.05 0.05 0.05 0.06 6 0.14 0.13 0.14 0.14 0.13 0.11 0.12 0.11 0.12 121.01 0.96 1.00 0.87 1.14 0.48 1.60 1.79 1.71 24 15.90 15.70 16.30 16.0514.60 0.13 3.96 8.54 11.10 36 15.10 17.45 16.80 18.75 22.15 0.09 3.028.69 16.55 48 15.95 16.90 19.25 21.10 24.00 0.07 2.05 8.10 16.50 7213.50 15.80 18.55 21.25 26.55 0.09 0.11 5.55 14.15 24 hours — — — —24.95 — — — — post- addition 48 hours — — — — 24.20 — — — — post-addition *Analysis from experiment #3.

TABLE 65 Ethanol Results Sample G132-100 Ethanol Concentration (g/L) 10%25% 50% 100% Sample 0% w/v w/v w/v w/v 0% 10% 25% 50% Time Xylose XyloseXylose Xylose Xylose Sugars* Sugars* Sugars* Sugars* 0 0.04 0.04 0.040.04 0.05 0.05 0.05 0.05 0.06 6 0.07 0.07 0.08 0.08 0.07 0.04 0.05 0.050.06 12 0.60 0.56 0.67 0.58 0.71 0.13 1.37 1.48 1.44 24 13.05 14.4514.90 13.95 12.05 0.03 3.67 7.62 10.55 36 15.10 17.10 18.25 18.20 19.250.01 3.09 8.73 16.10 48 14.40 17.00 19.35 22.55 24.45 0.01 1.91 7.7615.85 72 14.70 15.40 18.45 22.10 27.55 0.03 0.01 5.08 14.30 24 hours — —— — 25.20 — — — — post- addition 48 hours — — — — 24.60 — — — — post-addition *Analysis from experiment #3.

Samples were taken at 72 hours incubation for cell counts (see Tables66-67). Appropriately diluted samples were mixed with 0.05% Trypan blueand loaded into a Neubauer haemocytometer. The cells were counted under40× magnification.

Results

One seed flask was used to inoculate all Experiment #1 and #2 testflasks. The optical density (600 nm) of the seed flask was measured tobe 5.14 and the cell concentration was 4.65×10⁸ cells/mL (Tables 65-66).Therefore, the initial concentration of cells in the test flasks wasapproximately 4.65×10⁶ cells/mL.

A second seed flask was used to inoculate Experiment #3 flasks. Theoptical density (600 nm) of the seed flask was 5.78 and the cellconcentration was 3.75×10⁸ cells/mL. Therefore, the initialconcentration of cells in the test flasks was approximately 3.75×10⁶cells/mL.

TABLE 66 Cell Counts at Incubation Time of 72 hours Cell Concentration (×10⁸/mL) 0% 10% 25% 50% 100% 0% 10% 25% 50% Sample Xylose Xylose XyloseXylose Xylose Sugar Sugar Sugar Sugar A132-10 0.37 0.63 3.72 4.92 4.050.26 0.22 0.26 1.54 A132-100 0.99 1.07 0.99 0.78 1.97 0.03* 0.33 0.441.81 G132-10 0.95 4.50 2.67 2.67 3.82 0.01* 0.17 0.49 1.92 G132-100 6.534.02 4.84 4.47 5.29 0.01* 0.33 0.89 2.22 *Samples were heavilycontaminated after 72 hours of growth. This is expected because thePichia did not grow well without sugar added, and contaminants (from thenon-sterile samples) were able to out-grow the Pichia.

TABLE 67 Cell Counts at Incubation Time of 48 hours Post-Addition (100%Xylose and Glucose) Sample Cell Concentration (×10⁸/mL) A132-10 10.17A132-100 3.38 G132-10 3.94 G132-100 6.53

Example 31 Toxicity Testing of Lignocellulosic Samples Against P.stipitis and S. cerevisiae Summary

Thirty-seven samples were analyzed for toxicity against twoethanol-producing cultures, Saccharomyces cerevisiae and Pichiastipitis. In this study, glucose was added to the samples in order todistinguish between starvation of the cultures and toxicity of thesamples.

TABLE 68 Conditions for Toxicity Testing Organism Saccharomycescerevisiae Pichia stipitis Variable ATCC 24858 NRRL Y-7124 InoculationVolume (mL) 0.5-1 1 (target 6-7 × (target 3-4 × 10⁵ cells/mL) 10⁶cells/mL) Test Repetition Single Flasks Incubation Temperature 25° C.25° C. (±1° C.) Shaker Speed (rpm) 200 125 Type of Container 500 mL 250mL Erlenmeyer Flask Erlenmeyer Flask Media volume 100 mL 100 mL TotalIncubation time 72 72 (hours) Ethanol Analysis 0, 6, 12, 24, 0, 6, 12,24, (hours) 36, 48, 72 36, 48, 72 Cell Counts (hours) 24, 72 24, 72 pH 0hours 0 hours

Protocol

A summary of the protocol used is listed in Table 68. A description ofthe chemicals used in toxicity testing is listed in Table 69. Twocontrol flasks (no sample added) were performed for each microorganismfor each week of testing. A total of 82 flasks were analyzed.

During the experiments, no ethanol or cells appeared in the P. stipitisflasks containing samples C, C-1e, C-5e, and C-10e in the first 24 hoursof incubation. In order to confirm the results, the test was repeated.The second test confirmed some inhibition of P. stipitis growth whensamples C, C1E, C5E, and C10E were added to the flasks.

TABLE 69 Chemicals and Materials Used for Toxicity Testing MediaComponent Manufacturer Reference # Lot # Urea ScholAR Chemistry 9472706AD-7284-43 Yeast Nitrogen Becton Dickinson 291940 7128171 Base PeptoneBecton Dickinson 211677 4303198 Xylose Alfa Aesar A10643 10130919Glucose Sigma G-5400 107H0245 Yeast Extract Becton Dickinson 2886204026828 YM Broth Becton Dickinson 271120 6278265

TABLE 70 YSI Components Used in Toxicity Study Component Catalogue # YSIEthanol Membrane 2786 YSI Ethanol Standard (3.2 g/L) 2790 YSI EthanolBuffer 2787

Test Samples

Seven test samples (all with the C designation) were ground using acoffee grinder suitable for small samples. The samples were ground to aconsistent particle size (between samples) with the naked eye. Samplenumber C-100e ground easily to a small particle size.

All samples were added to the flasks at a concentration of 50 grams perliter with the exception of the six P samples (25 grams per liter).These samples were white to off-white in color and visually fluffy andthe flasks would not mix properly (not enough free liquid) at the 50grams per liter concentration. Samples S dissolved easily and could inthe future be added to the flasks at a higher concentration. Samples Aand G could be added at 100 grams per Liter in the future.

Testing was performed using the two microorganisms as described below.

Saccharomyces cerevisiae ATCC 24858 (American Type Culture Collection)

A working cell bank of S. cerevisiae ATCC 24858 was prepared from arehydrated lyophilized culture obtained from American Type CultureCollection. Cryovials containing S. cerevisiae culture in 15% v/vglycerol are stored at −75° C. A portion of the thawed working cell bankmaterial will be streaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH5.0) and incubated at 30° C. for 2 days. A 250 mL Erlenmeyer flaskcontaining 50 mL of medium (20 g/L glucose, 3 g/L yeast extract, and 5.0g/L peptone, pH 5.0) was inoculated with one colony from the YM plateand incubated for 24 hours at 25° C. and 200 rpm. After 23 hours ofgrowth, a sample was taken and analyzed for optical density (600 nm in aUV spectrophotometer) and purity (Gram stain). Based on these results,one flask (called the Seed Flask) with an OD of 9-15 and pure Gram stainwas to be used for inoculating the growth flasks. After 23 hours ofgrowth, the seed flask had a low OD (5.14) and cell count (1.35×10⁸cells/mL). Of note, the colony taken from the seed plate was smallerthan usual. Therefore, 0.5 mL of seed material (as opposed to theplanned 0.1 mL) was added to each test vessel.

The test vessels were 500 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved at 121° C.and 15 psi prior to the addition of the test materials. The testmaterials were not sterilized, as autoclaving would change the contentof the samples. The test samples were added at the time of inoculation(rather than prior to) to reduce the possibility of contamination. Inaddition to the test samples, 0.5-1.0 mL (0.5-1.0% v/v) of seed flaskmaterial was added to each flask. The flasks were incubated as describedabove for 72 hours.

Pichia stipitis (ARS Culture Collection)

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol are storedat −75° C. A portion of the thawed working cell bank material wasstreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for up to 5 days at 4° C.before use. A 250 mL Erlenmeyer flask containing 100 mL of medium (40g/L glucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/Lpeptone, 40 g/L xylose, pH 5.0) was inoculated with one colony andincubated for 24 hours at 25° C. and 125 rpm. After 23 hours of growth,a sample was taken and analyzed for optical density (600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, oneflask (called the Seed Flask) at an optical density of 5-9 and with apure Gram Stain was used to inoculate all of the test flasks.

The test vessels were 250 mL Erlenmeyer flasks containing 100 mL of thesterile medium described above. All flasks were autoclaved empty at 121°C. and 15 psi and filter sterilized (0.22 μm filter) medium added to theflasks prior to the addition of the test materials. The test materialswere not sterilized, as autoclaving would change the content of thesamples and filter sterilization not appropriate for sterilization ofsolids. The test samples were added at the time of inoculation (ratherthan prior to) to reduce the possibility of contamination. In additionto the test samples, 1 mL (1% v/v) of seed flask material was added toeach flask. The flasks were incubated as described above for 72 hours.

Analysis

Samples were taken from seed flasks just prior to inoculation and eachtest flask at 24 and 72 hours and analyzed for cell concentration usingdirect counts. Appropriately diluted samples of S. cerevisiae and P.stipitis were mixed with 0.05% Trypan blue, loaded into a Neubauerhaemocytometer. The cells were counted under 40× magnification.

Samples were taken from each flask at 0, 6, 12, 24, 36, 48 and 72 hoursand analyzed for ethanol concentration using the YSI Biochem Analyzerbased on the alcohol dehydrogenase assay (YSI, Interscience). Sampleswere centrifuged at 14,000 rpm for 20 minutes and the supernatant storedat −20° C. The samples will be diluted to 0-3.2 g/L ethanol prior toanalysis. A standard of 2.0 g/L ethanol was analyzed approximately every30 samples to ensure the integrity of the membrane was maintained duringanalysis.

Calculations

The following calculations were used to compare the cell counts andethanol concentration to the control flasks.

% performance=(concentration of ethanol in test flask/ethanol incontrol)*100%

cells=(number of cells in test flask/number of cells in controlflask)*100

Results

The S. cerevisiae seed flask had an optical density (600 nm) of 5.14 anda cell concentration of 1.35×10⁸ cells/mL. One half mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 6.75×10⁵/mL. During the second weekof testing, the S. cerevisiae seed flask had an optical density (600 nm)of 4.87 and a cell concentration of 3.15×10⁷ cells/mL. One mL of seedflask material was added to each of the test flasks. Therefore, thestarting cell concentration in each flask was 6.30×10⁵/mL. The pH of theS. cerevisiae flasks at a sample time of 0 hours is presented in Table71. The pH of the flask contents was within the optimal pH for S.cerevisiae growth (pH 4-6). No pH adjustment was required.

TABLE 71 pH of S. cerevisiae flasks at sample time 0 hours Sample SampleNumber pH Number pH P 5.04 C 5.46 P1E 4.99 C1E 5.54 P5E 5.04 C5E 5.50P10E 4.98 C10E 5.33 P50E 4.67 C30E 5.12 P100E 4.43 C50E 4.90 G 5.45C100E 4.66 G1E 5.47 ST 5.11 G5E 5.46 ST1E 5.06 G10E 5.39 ST5E 4.96 G50E5.07 ST10E 4.94 A 5.72 ST30E 5.68 A1E 5.69 ST50E 4.48 A5E 5.62 ST100E4.23 A10E 5.61 control A 5.02 A50E 5.74 control B 5.04 S* 5.10 S1E 5.08S5E 5.07 S10E 5.04 S30E 4.84 S50E 4.57 S100E 4.33 *“S” refers to sucrose*“C” refers to com *“ST” refers to starch

The ethanol concentration and performance in the S. cerevisiae flasksare presented in Table 72 and 73. The highest ethanol concentrationswere produced by the S (sucrose) samples.

TABLE 72 Ethanol Concentration in S. cerevisiae flasks Sample EthanolConcentration (g/L) at the following times (hours) Number 0 6 12 24 3648 72 P 0.02 0.04 0.38 5.87 7.86 5.41 1.04 P1E 0.03 0.03 0.28 5.10 8.035.46 0.58 P5E 0.03 0.04 0.57 8.84 6.38 3.40 0.04 P10E 0.06 0.05 0.656.63 7.66 5.57 1.40 P50E 0.04 0.03 0.26 2.80 5.85 8.59 5.68 P100E 0.040.02 0.12 3.64 8.26 7.51 3.03 G 0.04 0.04 0.57 10.20 8.24 6.66 2.84 G1E0.04 0.05 0.46 10.20 9.24 6.94 2.84 G5E 0.11 0.11 0.44 10.00 8.7 6.360.88 G10E 0.05 0.04 0.40 9.97 8.41 5.79 0.11 G50E 0.05 0.05 0.48 9.728.33 6.13 2.38 A 0.29 0.38 0.48 8.43 8.76 7.09 4.66 A1E 0.34 0.44 0.799.66 8.9 7.18 2.64 A5E 0.55 0.45 0.99 9.44 8.96 7.56 3.80 A10E 0.55 0.550.93 9.58 8.33 6.28 1.40 A50E 0.22 0.08 0.38 9.38 8.01 5.99 0.98 S 0.030.03 0.39 5.73 7.06 10.10 15.90 S1E 0.05 0.06 0.31 7.24 9.52 12.10 14.90S5E 0.02 0.05 0.34 5.87 7.68 11.90 19.00 S10E 0.03 0.04 0.35 5.88 7.7211.50 19.30 S30E 0.03 0.05 0.09 5.94 7.97 11.20 20.40 S50E* 0.13 0.190.47 5.46 7.96 13.00 18.30 S100E 0.11 0.10 0.21 7.00 10.6 13.80 12.70 C0.01 0.04 0.32 8.47 7.57 5.48 6.40 C1E 0.00 0.06 0.37 8.93 7.86 5.991.37 C5E 0.03 0.05 0.48 9.32 7.92 5.69 1.41 C10E 0.02 0.04 0.52 9.147.67 5.34 0.35 C30E 0.02 0.05 0.28 9.15 8.15 5.84 2.47 C50E 0.03 0.060.44 9.31 7.79 5.78 1.79 C100E 0.03 0.06 0.58 9.06 6.85 5.95 1.09 ST0.02 0.05 0.99 8.54 6.69 5.09 0.42 ST1E 0.03 0.04 0.70 8.87 7.29 4.811.04 ST5E 0.02 0.04 0.52 8.61 7.16 4.97 0.85 ST10E 0.02 0.05 0.33 8.977.05 5.26 0.68 ST30E 0.03 0.04 0.71 8.47 6.96 4.89 0.21 ST50E 0.04 0.070.34 8.46 8.19 7.04 3.20 ST100E 0.03 0.10 0.30 9.30 8.62 7.29 4.23control A 0.01 0.07 0.85 5.92 8.18 7.81 6.26 control B 0.01 0.04 0.274.86 6.43 8.01 6.75 control A* 0.04 0.21 1.36 5.19 7.31 7.55 5.16control B* 0.03 0.20 1.18 5.16 5.96 7.62 5.32 *analyzed week 2

TABLE 73 Performance in S. cerevisiae flasks Sample Performance (%) atthe following times (hours) Number 24 36 48 72 P 108.9 107.6 68.4 16.0P1E 94.6 109.9 69.0 8.9 P5E 164.0 87.3 43.0 0.6 P10E 123.0 104.9 70.421.5 P50E 51.9 80.1 108.6 87.3 P100E 67.5 113.1 94.9 46.5 G 189.2 112.884.2 43.6 G1E 189.2 126.5 87.7 43.6 G5E 185.5 119.1 80.4 13.5 G10E 185.0115.1 73.2 1.7 G50E 180.3 114.0 77.5 36.6 A 156.4 119.9 89.6 71.6 A1E179.2 121.8 90.8 40.6 A5E 175.1 122.7 95.6 58.4 A10E 177.7 114.0 79.421.5 A50E 174.0 109.7 75.7 15.1 S 106.3 96.6 127.7 244.2 S1E 134.3 130.3153.0 228.9 S5E 108.9 105.1 150.4 291.9 S10E 109.1 105.7 145.4 296.5S30E 110.2 109.1 141.6 313.4 S50E* 105.5 119.9 171.3 349.2 S100E 129.9145.1 174.5 195.1 C 157.1 103.6 69.3 98.3 C1E 165.7 107.6 75.7 21.0 C5E172.9 108.4 71.9 21.7 C10E 169.6 105.0 67.5 5.4 C30E 169.8 111.6 73.837.9 C50E 172.7 106.6 73.1 27.5 C100E 168.1 93.8 75.2 16.7 ST 158.4 91.664.3 6.5 ST1E 164.6 99.8 60.8 16.0 ST5E 159.7 98.0 62.8 13.1 ST10E 166.496.5 66.5 10.4 ST30E 157.1 95.3 61.8 3.2 ST50E 157.0 112.1 89.0 49.2ST100E 172.5 118.0 92.2 65.0 control A 109.8 112.0 98.7 96.2 control B90.2 88.0 101.3 103.7 control A* 100.3 110.1 99.5 98.5 control B* 99.799.7 100.4 101.5 *analyzed week 2

The cell concentration and % cells in the S. cerevisiae flasks arepresented in Table 74. High cell counts were observed in all flasks;however, not all of the cells appear to be making ethanol.

TABLE 74 S. cerevisea Cell Counts and % Cells Cell Count % Cells Sample(cells × 10⁸/mL) (count/count control) *100 Number 24 hours 72 hours 24hours 72 hour P 0.62 0.96 97.7 139.0 P1E 0.35 1.18 54.1 170.9 P5E 1.131.93 177.3 279.5 P10E 0.59 1.42 91.8 205.6 P50E 0.32 1.40 49.4 202.8P100E 0.45 1.94 70.6 281.0 G 0.74 3.48 116.5 504.0 G1E 0.68 3.65 107.1528.6 G5E 0.62 3.87 96.5 560.5 G10E 0.70 2.73 109.5 395.4 G50E 0.46 2.1071.8 304.1 A 0.55 3.53 86.0 511.2 A1E 0.83 3.45 130.7 499.6 A5E 0.673.53 104.8 511.2 A10E 0.53 1.95 83.6 282.4 A50E 0.66 1.62 103.5 234.6 S0.44 1.11 69.5 160.8 S1E 0.44 1.10 68.2 159.3 S5E 0.23 0.99 36.5 143.4S10E 0.39 0.73 61.2 105.4 S30E 0.31 0.71 48.3 102.1 S50E* 0.44 0.90 86.5196.5 S100E 0.53 0.84 82.4 121.7 C 0.45 1.81 70.6 262.1 C1E 0.71 2.40110.6 347.6 C5E 0.53 2.33 83.6 337.4 C10E 0.77 1.55 120.0 224.5 C30E0.75 1.80 117.6 260.7 C50E 0.64 1.70 100.1 246.2 C100E 0.81 1.51 127.1218.7 ST 0.75 1.75 117.6 253.4 ST1E 0.57 1.36 89.4 197.0 ST5E 0.58 1.4990.7 215.8 ST10E 0.61 1.32 95.4 191.2 ST30E 0.59 0.60 91.8 86.9 ST50E0.59 1.30 91.8 188.3 ST100E 0.41 1.24 63.5 179.6 control A 0.81 0.79127.1 114.1 control B 0.47 0.59 72.9 85.9 control A* 0.66 0.42 131.291.7 control B* 0.35 0.50 69.0 108.1

The P. stipitis seed flask had an optical density (600 nm) of 5.01 and acell concentration of 3.30×10⁸ cells/mL. One mL of seed flask materialwas added to each of the test flasks. Therefore, the starting cellconcentration in each flask was 3.30×10⁶/mL. During the second week oftesting, the P. stipitis seed flask had an optical density (600 nm) of5.45 and a cell concentration of 3.83×10⁸ cells/mL. One mL of seed flaskmaterial was added to each of the test flasks. Therefore, the startingcell concentration in each flask was 3.83×10⁶/mL. The pH of the P.stipitis flasks at a sample time of 0 hours is presented in Table 75.The pH of the flask contents was within the optimal pH for P. stipitisgrowth (pH 4-7). No pH adjustment was required.

TABLE 75 pH of P. stipitis Flasks at Sample Time 0 Hours Sample SampleNumber pH Number pH P 4.91 C 5.36 P1E 4.87 C1E 5.30 P5E 4.90 C5E 5.29P10E 4.78 C10E 5.06 P50E 4.46 C30E 4.89 P100E 4.24 C50E 4.70 G 5.45C100E 4.59 G1E 5.43 ST 4.93 G5E 5.48 ST1E 4.90 G10E 5.32 ST5E 4.81 G50E4.99 ST10E 4.83 A 5.69 ST30E 4.91 A1E 5.66 ST50E 4.24 A5E 5.60 ST100E4.07 A10E 5.58 control A 4.93 A50E 5.69 control B 4.91 S 5.00 S1E 4.94S5E 4.86 S10E 4.78 S30E 4.51 S50E 4.27 S100E 4.08

The ethanol concentration and performance in the P. stipitis flasks arepresented in Table 76 and 77. The highest ethanol concentrations werethe G and A series. Flasks C-30e, C-50e, and C-100e also contained highconcentrations of ethanol. The cell concentration and % cells in the P.stipitis flasks are presented in Table 78. Low cell concentrations wereobserved in the flasks with the S designations. Low cell counts werealso observed in flasks containing samples C, C1E, C5E, and C10E at the24 hour sample time.

TABLE 76 Ethanol concentration in P. stipitis flasks Sample EthanolConcentration (g/L) at the following times (hours) Number 0 6 12 24 3648 72 P 0.01 0.05 0.26 4.98 8.57 14.10 17.00 P1E 0.02 0.03 0.04 4.249.03 12.40 17.30 P5E 0.02 0.03 0.42 6.72 12.40 15.60 18.60 P10E 0.020.02 0.01 1.38 8.69 13.00 17.00 P50E 0.01 0.02 0.02 0.03 3.77 10.5016.90 P100E 0.02 0.03 0.02 3.75 10.50 15.60 18.80 G 0.02 0.08 0.20 10.8017.70 19.40 25.40 G1E 0.04 0.12 0.50 12.20 19.60 23.80 28.60 G5E 0.070.14 0.73 12.50 19.10 24.50 27.50 G10E 0.04 0.19 0.42 10.20 19.10 22.9028.20 G50E 0.05 0.22 0.25 8.73 18.40 22.20 28.00 A 0.13 0.28 0.82 16.1019.40 19.30 18.60 A1E 0.22 0.59 1.08 16.10 22.40 27.60 27.70 A5E 0.320.43 0.43 10.60 22.10 27.10 28.10 A10E 0.33 0.61 1.15 14.90 22.00 27.1027.90 A50E 0.30 0.10 0.47 13.40 20.20 24.80 27.10 S 0.01 0.01 0.26 3.687.50 10.20 13.30 S1E 0.02 0.02 0.22 4.98 9.22 11.60 14.20 S5E 0.02 0.020.19 4.25 8.50 11.70 14.70 S10E 0.03 0.02 0.17 2.98 8.87 11.90 14.70S30E 0.08 0.05 0.03 2.96 8.73 12.60 16.50 S50E 0.08 0.05 0.04 2.24 6.137.95 12.50 S100E 0.11 0.10 0.08 3.36 7.82 10.50 13.90 C* 0.02 0.03 0.050.23 1.66 2.68 6.57 C1E* 0.03 0.03 0.03 0.07 0.95 1.85 10.20 C5E* 0.030.02 0.04 0.05 0.37 1.59 4.80 C10E* 0.03 0.04 0.04 0.05 3.91 15.20 28.30C30E 0.01 0.03 0.60 12.30 21.20 26.00 27.20 C50E 0.02 0.02 0.45 12.3019.50 23.80 29.20 C100E 0.05 0.04 0.38 11.40 18.70 22.90 27.70 ST 0.030.03 0.37 6.69 10.70 13.50 10.90 ST1E 0.01 0.00 0.48 5.24 9.37 12.5015.70 ST5E 0.02 0.03 0.29 5.45 10.10 11.90 14.70 ST10E 0.02 0.02 0.425.60 9.44 12.20 14.90 ST30E 0.05 0.04 0.73 5.70 9.50 12.10 15.20 ST50E0.02 0.05 0.19 5.16 9.47 12.70 15.20 ST100E* 0.07 0.15 0.11 4.98 10.7015.40 18.80 control A 0.02 0.03 0.37 4.05 7.50 9.24 11.50 control B 0.020.02 0.30 4.22 7.44 9.44 11.50 Control A* 0.02 0.05 0.69 4.86 8.69 11.1016.40 Control B* 0.02 0.05 0.74 5.96 10.80 13.00 14.00 *analyzed week 2

TABLE 77 Performance in P. stipitis flasks Sample Performance (%) at thefollowing times (hours) Number 24 36 48 72 P 120.3 114.7 151.0 147.8 P1E102.4 120.9 132.8 150.4 P5E 162.3 166.0 167.0 161.7 P10E 33.3 116.3139.2 147.8 P50E 0.7 50.5 112.4 147.0 P100E 90.6 140.6 167.0 163.5 G260.9 236.9 207.7 220.9 G1E 294.7 262.4 254.8 248.7 G5E 301.9 255.7262.3 239.1 G10E 246.4 255.7 245.2 245.2 G50E 210.9 246.3 237.7 243.5 A388.9 259.7 206.6 161.7 A1E 388.9 299.9 295.5 240.9 A5E 256.0 295.9290.1 244.3 A10E 359.9 294.5 290.1 242.6 A50E 323.7 270.4 265.5 235.7 S88.9 100.4 109.2 115.7 S1E 120.3 123.4 124.2 123.5 S5E 102.7 113.8 125.3127.8 S10E 72.0 118.7 127.4 127.8 S30E 71.5 116.9 134.9 143.5 S50E 54.182.1 85.1 108.7 S100E 81.2 104.7 112.4 120.9 C* 4.2 17.0 22.2 43.2 C1E*1.4 9.7 15.4 67.1 C5E* 0.9 3.8 13.2 31.6 C10E* 0.9 40.1 126.1 246.1 C30E297.1 283.8 278.4 236.5 C50E 297.1 261.0 254.8 253.9 C100E 275.4 250.3245.2 240.9 ST 161.6 143.2 144.5 94.8 ST1E 126.6 125.4 133.8 136.5 ST5E131.6 135.2 127.4 127.8 ST10E 135.3 126.4 130.6 129.6 ST30E 137.7 127.2129.6 132.2 ST50E 124.6 126.8 136.0 132.2 ST100E* 120.3 109.7 127.8123.7 control A 97.8 100.4 98.9 100.0 control B 101.9 99.6 101.1 100.0control A* 89.8 89.1 92.1 107.9 control B* 110.2 110.8 107.9 92.1*analyzed in week 2

TABLE 78 P. stipitis Cell Counts and % Cells Cell Count % Cells Sample(cells × 10⁸/mL) (count/count control) *100 Number 24 hours 72 hours 24hours 72 hours P 2.78 11.00 80.6 148.0 P1E 2.10 7.20 60.9 96.9 P5E 2.939.68 84.9 130.3 P10E 1.42 7.73 41.2 104.0 P50E 0.33 8.63 9.6 116.2 P100E1.58 8.25 45.8 111.0 G 1.50 14.20 43.5 191.1 G1E 3.90 8.10 113.0 109.0G5F 2.93 6.45 84.9 86.8 G10E 4.35 13.30 126.1 179.0 G50E 3.75 11.60108.7 156.1 A 7.43 8.55 215.4 115.1 A1E 4.13 9.53 119.7 128.3 A5E 3.689.75 106.7 131.2 A10E 4.50 7.50 130.4 100.9 A50E 6.23 5.33 180.6 71.7 S3.53 5.55 102.3 74.7 S1E 3.00 3.30 87.0 44.4 S5E 3.68 3.00 106.7 40.4S10E 1.73 5.78 50.1 77.8 S30E 2.55 5.48 73.9 73.8 S50E 2.63 6.15 76.282.8 S100E 2.25 4.43 65.2 59.6 C* 0.00 0.26 0.00 7.2 C1E* 0.00 0.36 0.009.9 C5E* 0.00 0.08 0.00 2.1 C10E* 0.00 5.85 0.00 160.7 C30E 5.78 4.20167.5 56.5 C50E 3.40 7.35 98.6 98.9 C100E 1.98 6.60 57.4 88.8 ST 2.557.65 73.9 103.0 ST1E 2.00 8.70 58.0 117.1 ST5E 1.85 6.75 53.6 90.8 ST10E1.83 5.40 53.0 72.7 ST30E 2.78 6.15 80.6 82.8 ST50E 1.33 3.45 38.6 46.4ST100E* 4.35 3.83 59.8 105.2 control A 3.60 7.13 104.3 96.0 control B3.30 7.73 95.7 104.0 control A* 7.50 3.23 103.0 88.7 control B* 7.054.05 96.8 111.3 *analyzed week 2

Cell Toxicity Results Summary

Zymomonas mobilis

As shown in FIG. 65A, elevated cell numbers (e.g., greater than thecontrol) were observed in samples containing P-132-10, G-132-10, andWS-132-10 at the 24 hour time point. Cell numbers in the presence of allother samples were comparable to the control. This observation indicatesthat the substrates were not toxic towards Z. mobilis for up to 24 hoursafter seeding.

At the 36 hour time point, a decrease in cell numbers (e.g., due to aloss of cells or cell death) was observed for all samples, including thecontrol. The greatest decrease in cell numbers was observed for thosesamples containing P-132-10, G-132-10. The likely cause of this effectis common to all samples, including the control. Thus, the cause of thiseffect is not the test substrates, as these vary in each sample, and arenot present in the control. Possible explanations for this observationinclude inappropriate culture conditions (e.g., temperature, mediacompositions), or ethanol concentrations in the sample.

As shown in FIG. 65B, all cells produced comparable amounts of ethanol(e.g., 5-10 g/L) at each time point, irrespective of the substrate.Consistent with the cell number data presented in FIG. 65A, ethanolconcentration in each sample peaked at the 24 hour time point. Incontrast to the cell number data, ethanol concentration did not decreaseat subsequent time points. This was expected as ethanol was not removedfrom the system. In addition, this data suggests that ethanol productionin these samples may have resulted from fermentation of glucose in theculture media. None of the substrates tested appeared to increaseethanol production.

Together, FIGS. 65A and 65B suggest that ethanol concentrations aboveabout 6 g/L may be toxic to Z. mobilis. This data is also presented as apercentage normalized against the control, as shown in FIG. 65C.

Pichia stipitis

As shown in FIG. 66, cell numbers were comparable to the control.Furthermore, although slightly reduced cell numbers were present insamples containing G-132 and WS-132, reduced cell numbers were notobserved for G-132-10, G-132-100, A-132-10, or A-132-100. Thus, it isunlikely that substrates G or A are toxic. Rather, the reduced cellnumbers observed for G-132 and WS-132 are likely to have been caused byan experimental anomaly or by the presence of unprocessed substratesomehow impeding cell growth. Overall, this data suggests that glucosepresent in the control and experimental samples is likely to besufficient to promote optimal P. stipitis growth, and that the presenceof an additional substrate in the sample does not increase this growthrate. These results also suggest that none of the samples are toxic inP. stipitis.

As shown in FIG. 66A, despite the similar cell numbers reported in FIG.66A, greatly increased ethanol production was observed in all samplescontaining an experimental substrate. Ethanol concentrations increasedover time for each of the three time points tested. The highestconcentration of ethanol was observed for A-132-10 at the 48 hour timepoint (e.g., approximately 26.0 g/L). By comparing the substrateconcentrations with the highest levels of ethanol production with thecell number data presented in FIG. 66A, it can be seen that P. stipitisdo not appear to be sensitive to increasing ethanol concentrations.Furthermore, ethanol production does not appear to be related to cellnumber, but rather appears to be related to the type of substratepresent in the sample.

Together, the results presented in FIGS. 66 and 66A suggest that theexperimental substrates do not promote increased P. stipitis growth,however, they greatly increase the amount of ethanol produced by thiscell type. This data is also presented as a percentage normalizedagainst the control, as shown in FIG. 66B.

Saccharomyces cerevisiae

As shown in FIG. 67, G-132-100, A-132, A-132-10, A-132-100, and WS-132promoted slightly elevated cell numbers compared to the control. Nosignificant reductions in cell number were observed for any sample.These results suggest that none of the samples are toxic in S.cerevisiae.

As shown in FIG. 67A, increased ethanol production was observed in cellstreated with each cell type compared to the control. Comparison of thosesamples containing the highest amount of ethanol with the cell numberdata presented in FIG. 67 suggests that ethanol concentrations in excessof 5 g/L may have had an adverse effect on cell numbers. However, thisobservation is not the case for all samples.

This data is also presented as a percentage normalized against thecontrol, as shown in FIG. 67B.

In conclusion, none of the samples tested appeared to be toxic in Z.mobilis, P. stipitis, and S. cerevisiae. Furthermore, P. stipitisappeared to be the most efficient of the three cell types for producingethanol from the experimental substrates tested.

Example 32 Shake Flask Fermentation Studies Using P. stipitis Summary

Shake flask fermentation studies using various enzymes, physicaltreatments, and Pichia stipitis were performed.

Protocol

Experiments were performed under the parameters outlined in Table 79.

TABLE 79 Chemicals and Materials Used for the Shake Flask ExperimentMedia Component Manufacturer Reference # Urea ScholAR 9472706 ChemistryYeast Nitrogen Becton 291940 Base Dickinson Peptone Becton 211677Dickinson Xylose Alfa Aesar A10643 Glucose Sigma G-5400 Yeast ExtractBecton 288620 Dickinson YM Broth Becton 271120 Dickinson NOVOZYME ® 188Novozymes Sigma #C6105 Celluclast 1,5 FG Novozymes Sigma #C2730 SolkaFloc International 200 NF Fibre Corporation Pluronic F-68 Sigma P1300ACCELLERASE ® 1000 Genencor N/A

Seed Development

A working cell bank of P. stipitis NRRL Y-7124 was prepared from arehydrated lyophilized culture obtained from ARS Culture Collection.Cryovials containing P. stipitis culture in 15% v/v glycerol were storedat −75° C. A portion of the thawed working cell bank material werestreaked onto a Yeast Mold (YM) Broth+20 g/L agar (pH 5.0) and incubatedat 30° C. for 2 days. The plates were held for up to seven days at 4° C.before use.

A 250 mL Erlenmeyer flask containing 100 mL of medium (40 g/L glucose,1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, 40 g/Lxylose, pH 5.0) were inoculated with one colony and incubated for 24hours at 25° C. and 150 rpm. After 23 hours of growth, a sample wastaken and analyzed for optical density (OD 600 nm in a UVspectrophotometer) and purity (Gram stain). Based on these results, twoflasks (called the Seed Flask I at an OD of between 4 and 8 and with aclean Gram stain was combined to inoculate the growth flasks.

Exemplary Experiments

Experiments were performed to 1) determine the correct sonifier outputand temperature regulation (below 60° C.) and 2) confirm theconcentration of Celluclast 1,5 FG and Novozyme 188 with and withoutPluronic F-68.

Five hundred milliliters of water were added to a 1 L glass beaker. Thehorn of a Branson Model 450 Sonifier was placed ½ inch into the surfaceof the beaker and set at a maximum constant output for 60 minutes. Thetemperature of the water was measured every 10 minutes for 60 minutes ofsonication.

An experiment was performed to determine if 1) the concentration ofCelluclast 1,5 FG and Novozyme 188 (0.5 mL and 0.1 mL per gram ofbiomass, respectively) was sufficient for the shake flask experimentsand 2) if the addition of Pluronic F68 augmented the hydrolysis ofcellulose. Four 250 mL flasks were prepared with 100 mL of sterile broth(1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, pH 5.0).Duplicate flasks contained 1% w/v Pluronic F-68. Solka Floc CrystallineCellulose (6 g) was added to the flasks and allowed to soak at roomtemperature for 14 hours. Celluclast 1,5 FG and Novozyme 188 (0.5 mL and0.1 mL per gram of Solka Floc, respectively) were added and each flaskincubated at 50° C. for 24 hours at 100 rpm. Samples were taken prior tothe addition of enzyme and 24 hours post enzyme addition from all fourflasks and analyzed for glucose concentration using the YSI BiochemAnalyzer (YSI, Interscience). One milliliter of Pichia stipitis seedflask contents was added to the four flasks and incubated at 25° C. and125 rpm for 24 hours. Samples were taken from each flask prior toinoculation and after 24 hours incubation and analyzed for ethanolconcentration using the YSI Biochem Analyzer (YSI, Interscience).

Test Flasks

The test flasks were 2.8 L Fernbach flasks holding 900 mL of broth (1.7g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone, pH 5.0).Control flasks were 250 mL flasks containing 100 mL of broth (40 g/Lglucose, 1.7 g/L yeast nitrogen base, 2.27 g/L urea, 6.56 g/L peptone,40 g/L xylose, pH 5.0). The exact nature of each flask was decided byXyleco and is described in Table 80 below.

Samples were not sterilized prior to the start of the experiment. Allsamples were added to the flasks and allowed to soak for 15 hours atroom temperature. Some of the samples were sonicated for one hour usinga Branson Model 450 Sonifier equipped with a ½ inch disruptor horn. Theoriginal plan was to split the flask contents into two, and sonicateeach half continuously at the maximum output for the equipment up to 450watts, (the allowable output depends on the viscosity of the sample) for1 hour. An output setting of 3 and a Duty cycle of Pulse 90% weresufficient for the mixing of the beaker contents. At an output settingof 3, the meter read between 30 and 40. The output was calculated to be40-60 watts.

Originally, the plan was to mix some samples (see Table 80) for varioustimes using a POLYTRON PT 10/35 laboratory homogenizer (or rotor/stator)at 25,000 rpm for various times. Samples #22 and #23 were split into twobeakers and treated for 30 minutes using the large Kinematica PolytronPT 10/35. The generator (tip) was a PTA 20 with a stator diameter of 20mm. The instrument was operated at a speed of 11,000 rpm. Operationabove 11,000 rpm caused splattering of beaker contents, movement of thebeaker, and over-heating of the equipment. After samples #23 and #24,the Polytron PT 10/35 stopped working, presumably from over-use withquite viscous samples. Therefore, the hand-held Polytron PT1200C wasused. The generator (tip) was a PT-DA 1212 with a stator diameter of 12mm. The instrument could be operated at 25,000 rpm. It was noted by theoperator that a similar degree of mixing was observed with the hand-heldat 25,000 rpm as compared to the larger model at 11,000 rpm. The samplewas periodically mixed by the operator to ensure even mixing. Samples 19through 22 were mixed with the hand-held Polytron PT1200C.

Enzyme pretreatments included: 1) E1=ACCELLERASE® 1000 at a loadingdensity of 0.25 mL per gram of substrate and 2) E2=Celluclast 1,5 FG andNovozyme 188 at a loading concentration of 0.5 and 0.1 mL per gram ofsubstrate, respectively. After physical pretreatment (see Table 80below), the appropriate enzyme(s) were added and the flasks held at 50°C. and 125 rpm for 20 hours. After 20 hours, the flasks were cooled toroom temperature for 1 hour prior to the addition of P. stipitis.

TABLE 80 Summary of Test Treatments Sample Enzyme Concen- TreatmentSample tration Physical (50° C., Test Number Number (g/900 mL Treatment21 hours Control (250 mL None — — — flask performed in duplicate eachweek Week 1 1 SP 35 15 h r.t. soak None 2 XP 35 15 h r.t. soak None 3 SP35 15 h r.t. soak E1 4 SP 35 15 h r.t. soak E2 5 XP 35 15 h r.t. soak E16 XP 35 15 h r.t. soak E2 7 XP-10e 35 15 h r.t. soak E2 8 XP-30e 35 15 hr.t. soak E2 9 XP-50e 35 15 h r.t. soak E2 10 XP-10e 35 15 h r.t. soak,1 E2 hour sonication 11 XP-30e 35 15 h r.t. soak, 1 E2 hour sonication12 XP-50e 35 15 h r.t. soak, 1 E2 hour sonication Week 2 13 XP-10e 35 15h r.t. soak, 10 E2 min sonication 14 XP-30e 35 15 h r.t. soak, 10 E2 minsonication 15 XP-50e 35 15 h r.t. soak, 10 E2 min sonication 16 XP-10e35 15 h r.t. soak, 30 E2 min sonication 17 XP-30e 35 15 h r.t. soak, 30E2 min sonication 18 XP-50e 35 15 h r.t. soak, 30 E2 min sonication 19XP-10e 35 15 h r.t. soak, 10 E2 min rotor/stator 20 XP-30e 35 15 h r.t.soak, 10 E2 min rotor/stator 21 XP-50e 35 15 h r.t. soak, 10 E2 minrotor/stator 22 XP-10e 35 15 h r.t. soak, 30 E2 min rotor/stator 23XP-30e 35 15 h r.t. soak, 30 E2 min rotor/stator 24 XP-50e 35 15 h r.t.soak, 30 E2 min rotor/stator

Analysis

A sample was taken from each flask after physical and/or enzymepretreatment (just prior to the addition of P. stipitis) and analyzedfor glucose concentration using the YSI Biochem Analyzer (YSI,Interscience). Samples were centrifuged at 14,000 rpm for 20 minutes andthe supernatant stored at −20° C. The samples were diluted to between0-25.0 g/L glucose prior to analysis. A glucose standard was analyzedapproximately every 30 samples to ensure the integrity of the membranewas maintained.

A total of five samples were taken from each flask at 0, 12, 24, 48, and72 hours and analyzed for ethanol concentration using the YSI BiochemAnalyzer based on the alcohol dehydrogenase assay (YSI, Interscience).Samples were centrifuged at 14,000 rpm for 20 minutes and thesupernatant stored at −20° C. and diluted to between 0-3.0 g/L ethanolprior to analysis. A standard of 2.0 g/L ethanol was analyzedapproximately every 30 samples to ensure the integrity of the membranewas maintained.

A sample of the seed flask was analyzed in order to determine theinitial cell concentration in the test flasks. In addition one sample at72 hours of incubation was taken from each flask and analyzed for cellconcentration. Appropriately diluted samples were mixed with 0.05%Trypan blue and loaded into a Neubauer haemocytometer. The cells werecounted under 40× magnification.

Results Experiments

The results of a sonifier experiment are presented in Table 81. Therewere no problems with over-heating of the water.

TABLE 81 Sonifier Experiment Time Temperature (° C.) 0 18 10 18 20 19 3019 40 19 50 19 60 19

The results of the experiment to confirm the concentration of Celluclast1,5 FG and Novozyme 188 with and without Pluronic F-68 are presented inTable 82 and 83. A concentration of 60 g/L cellulose (Solka Floc) wasadded to each flask. After 24 hours of incubation, 33.7 to 35.7 g/Lglucose was generated (30.3 to 32.1 g/L cellulose digested).

After 24 hours of incubation with P. stipitis, 23.2-25.7 g/L of glucoseremained in the flasks. This indicates that not all of the glucose wasused within 24 hours of incubation.

There was no evidence of Pluronic F-68 toxicity toward P. stipitis.However, there was no increase in the amount of glucose generated aftera 24 hour enzyme treatment with the addition of Pluronic F-68.

TABLE 82 Glucose Results Glucose Concentration (g/L) Prior After EnzymeAfter to Enzyme Treatment (50° C., P. stipitis Flask Treatment 24 hours,100 rpm for 24 hours Control A 0.28 34.3 23.2 Control B 0.64 35.7 25.3Containing Pluronic A 0.48 34.8 25.6 Containing Pluronic B 0.93 33.725.7

TABLE 83 Ethanol Results Ethanol Concentration (g/L) at times (hours) 0(inoculation, 24 hours of Flask after enzyme treatment) P. stipitisControl A 0.01 7.23 Control B 0.01 5.75 Containing Pluronic A 0.01 7.57Containing Pluronic B 0.00 7.36

During week one of testing, the seed flask had an optical density (600nm) of 9.74 and a cell concentration of 4.21×10⁸ cells/mL. Nine mL ofseed flask material was added to each of the test flasks and 1 mL to thecontrol flasks (1% v/v). Therefore, the starting cell concentration ineach flask was ×4.21×10⁶/mL.

During week two of testing, the seed flask had an optical density (600nm) of 3.02 and a cell concentration of 2.85×10⁸ cells/mL. To accountfor differences in cell counts and OD, 12 mL of seed flask material wasadded to each of the test flasks and 1.5 mL to the control flasks (1.5%v/v). Therefore, the starting cell concentration in each flask was3.80×10⁶/mL.

The ethanol concentration in the flasks is presented in Table 84. Thehighest concentration of ethanol was observed in Flask #6 (Sample XP,Overnight Soak, treatment with E2 at 50° C. for 21 hours). Aconcentration of 19.5 g/L (17.55 g/per flask) was generated from anoriginal 35 grams of substrate in 48 hours. The yield of ethanol (gramsof ethanol/gram of substrate) in flask #6 was 0.50.

TABLE 84 Ethanol Concentration Sample Ethanol Concentration (g/L) atIncubation Time (hours) Number 0 12 24 48 72 Control A 0.249 1.57 9.3113.60 14.20 Control B 0.237 1.04 7.97 11.40 13.90 1 0.247 0.16 0.10 0.110.06 2 0.175 0.12 0.10 0.17 0.29 3 0.284 2.73* 8.88 9.72 10.40 4 0.3980.43 8.02 14.40 12.10 5 0.312 0.31 10.30 11.30 18.80 6 0.399 0.73 7.5519.50* 19.00 7 0.419 0.38 4.73 16.80* 15.40 8 0.370 0.46 0.56 9.86 13.509 0.183 0.47 0.53 12.00 14.10 10 0.216 0.35 6.11 13.80 15.60 11 0.1990.33 0.88 9.02 8.52 12 0.264 0.43 0.41 8.76 13.80 Control A 0.49 0.847.93 13.00 15.00 Control B 0.50 0.93 8.39 13.40 15.00 13 0.86 0.99 8.4210.50 14.20 14 0.95 0.88 3.79 10.90 12.40 15 1.18 0.42 1.12 9.26 12.6016 0.88 0.42 5.41 6.78 12.80 17 0.99 0.45 1.73 10.60 12.00 18 1.17 0.461.12 10.60 12.10 19 0.78 0.50 9.75 12.60 13.40 20 0.94 0.39 2.54 11.1012.20 21 1.28 0.43 1.46 11.50 11.30 22 0.84 1.09 10.00 14.00 10.10 230.96 0.57 6.77 11.10 12.10 24 1.20 0.42 1.91 12.10 13.10 *Samplesanalyzed twice with the same result. Flasks with a concentration ofgreater than 15 g/L ethanol are in BOLD.

The results of the glucose analysis are presented in Table 85. After 21hours of enzyme treatment, the highest concentration of glucose was 19.6g/L (17.6 grams per flask) in flask #6 (Sample XP, Overnight Soak,treatment with E2 at 50° C. for 21 hours). This was also the flask withthe highest ethanol concentration (see Table 84). After 72 hours, verylittle glucose remained in the flasks. No glucose was detected in Flasks1 and 2.

TABLE 85 Glucose Concentration Glucose Concentration (g/L) at SampleIncubation Time (hours) Number 0 72 1 0.0 0.00 2 0.0 0.00 3 7.2 0.02 413.3 0.03 5 15.9 0.05 6 19.6 0.05 7 13.9 0.04 8 15.4 0.06 9 18.3 0.09 1017.1 0.05 11 13.0 0.04 12 17.0 0.08 13 14.4 0.03 14 13.7 0.04 15 16.30.08 16 13.2 0.03 17 13.4 0.04 18 15.8 0.06 19 15.3 0.04 20 14.3 0.04 2115.5 0.06 22 14.7 0.04 23 13.5 0.04 24 16.6 0.07

The results of the direct cell counts are presented in Table 86. Theconcentration of viable cells was higher in the control flasks. Thelowest counts were observed in flasks 1 through 4.

TABLE 86 Cell Counts Sample Number of Cells (×106/mL) after Number 72hours of incubation Control A 38.30 Control B 104.00 1 0.02 2 0.08 30.07 4 0.06 5 0.15 6 1.05 7 1.50 8 1.95 9 1.05 10 3.60 11 1.28 12 0.90Control A 39.80 Control B 30.80 13 0.98 14 0.40 15 0.63 16 0.71 17 1.1518 0.83 19 1.25 20 1.02 21 0.53 22 0.56 23 0.59 24 0.59

Example 32 Alcohol Production Using Irradiation-Sonication Pretreatment

The optimum size for biomass conversion plants is affected by factorsincluding economies of scale and the type and availability of biomassused as feedstock. Increasing plant size tends to increase economies ofscale associated with plant processes. However, increasing plant sizealso tends to increase the costs (e.g., transportation costs) per unitof biomass feedstock. Studies analyzing these factors suggest that theappropriate size for biomass conversion plants can range from 2000 to10,000 dried tons of biomass feedstock per day. The plant describedbelow is sized to process 2000 tons of dry biomass feedstock per day.

FIG. 39 shows a process schematic of a biomass conversion systemconfigured to process switchgrass. The feed preparation subsystemprocesses raw biomass feedstock to remove foreign objects and provideconsistently sized particles for further processing. The pretreatmentsubsystem changes the molecular structure (e.g., reduces the averagemolecular weight and the crystallinity) of the biomass feedstock byirradiating the biomass feedstock, mixing the irradiated the biomassfeedstock with water to form a slurry, and applying ultrasonic energy tothe slurry. The irradiation and sonication convert the cellulosic andlignocellulosic components of the biomass feedstock into fermentablematerials. The primary process subsystem ferments the glucose and otherlow weight sugars present after pretreatment to form alcohols.

Example 32 Proton Irradiation of Cellulosic Material

The fibrous material of Example 4 is irradiated in vacuum with a beam ofprotons from a tandem PELLERTRON® accelerator 5SDH-2 (NationalElectrostatics Corporation). The energy of each proton and the currentdensity can range from 2.0 to 3.12 MeV and from 0.3 to 140 nA/cm²,respectively, which corresponds to a fluence rate of about 10⁹-10¹² cm⁻²s⁻¹. Molecular weight breakdown can start to occur from about 1 Mrad.Cross-linking can occur below this level.

Feed Preparation

The selected design feed rate for the plant is 2,000 dry tons per day ofswitchgrass biomass. The design feed is chopped and/or shearedswitchgrass.

Biomass feedstock in the form of bales of switchgrass is received by theplant on truck trailers. As the trucks are received, they are weighedand unloaded by forklifts. Some bales are sent to on-site storage whileothers are taken directly to the conveyors. From there, the bales areconveyed to an automatic unwrapping system that cuts away any plasticwrapping and/or net surrounding the bales. The biomass feedstock is thenconveyed past a magnetic separator to remove tramp metal, after which itis introduced to shredder-shearer trains where the material is reducedin size. Finally, the biomass feedstock is conveyed to the pretreatmentsubsystem.

In some cases, the switchgrass bales are wrapped with plastic net toensure they don't break apart when handled, and may also be wrapped inplastic film to protect the bale from weather. The bales are eithersquare or round. The bales are received at the plant from off-sitestorage on large truck trailers.

Since switchgrass is only seasonally available, long-term storage isrequired to provide feed to the plant year-round. Long-term storage willlikely consist of 400-500 acres of uncovered piled rows of bales at alocation (or multiple locations) reasonably close to the ethanol plant.On-site short-term storage is provided equivalent to 72 hours ofproduction at an outside storage area. Bales and surrounding access waysas well as the transport conveyors will be on a concrete slab. Aconcrete slab is used because of the volume of traffic required todeliver the large amount of biomass feedstock required. A concrete slabwill minimize the amount of standing water in the storage area, as wellas reduce the biomass feedstock's exposure to dirt. The stored materialprovides a short-term supply for weekends, holidays, and when normaldirect delivery of material into the process is interrupted.

The bales are off-loaded by forklifts and are placed directly onto baletransport conveyors or in the short-term storage area. Bales are alsoreclaimed from short-term storage by forklifts and loaded onto the baletransport conveyors.

Bales travel to one of two bale unwrapping stations. Unwrapped bales arebroken up using a spreader bar and then discharged onto a conveyor whichpasses a magnetic separator to remove metal prior to shredding. A trampiron magnet is provided to catch stray magnetic metal and a scalpingscreen removes gross oversize and foreign material ahead of multipleshredder-shearer trains, which reduce the biomass feedstock to theproper size for pretreatment. The shredder-shearer trains includeshredders and rotary knife cutters. The shredders reduce the size of theraw biomass feedstock and feed the resulting material to the rotaryknife cutters. The rotary knife cutters concurrently shear the biomassfeedstock and screen the resulting material.

Three storage silos are provided to limit overall system downtime due torequired maintenance on and/or breakdowns of feed preparation subsystemequipment. Each silo can hold approximately 55,000 cubic feet of biomassfeedstock (˜3 hours of plant operation).

Pretreatment

A conveyor belt carries the biomass feedstock from the feed preparationsubsystem 110 to the pretreatment subsystem 114. As shown in FIG. 40, inthe pretreatment subsystem 114, the biomass feedstock is irradiatedusing electron beam emitters, mixed with water to form a slurry, andsubjected to the application of ultrasonic energy. As discussed above,irradiation of the biomass feedstock changes the molecular structure(e.g., reduces the average molecular weight and the crystallinity) ofthe biomass feedstock. Mixing the irradiated biomass feedstock into aslurry and applying ultrasonic energy to the slurry further changes themolecular structure of the biomass feedstock. Application of theradiation and sonication in sequence may have synergistic effects inthat the combination of techniques appears to achieve greater changes tothe molecular structure (e.g., reduces the average molecular weight andthe crystallinity) than either technique can efficiently achieve on itsown. Without wishing to be bound by theory, in addition to reducing thepolymerization of the biomass feedstock by breaking intramolecular bondsbetween segments of cellulosic and lignocellulosic components of thebiomass feedstock, the irradiation may make the overall physicalstructure of the biomass feedstock more brittle. After the brittlebiomass feedstock is mixed into a slurry, the application of ultrasonicenergy further changes the molecular structure (e.g., reduces theaverage molecular weight and the crystallinity) and also can reduce thesize of biomass feedstock particles.

Electron Beam Irradiation

The conveyor belt 491 carrying the biomass feedstock into thepretreatment subsystem distributes the biomass feedstock into multiplefeed streams (e.g., 50 feed streams) each leading to separate electronbeam emitters 492. In this embodiment, the biomass feedstock isirradiated while it is dry. Each feed stream is carried on a separateconveyor belt to an associated electron beam emitter. Each irradiationfeed conveyor belt can be approximately one meter wide. Before reachingthe electron beam emitter, a localized vibration is induced in eachconveyor belt to evenly distribute the dry biomass feedstock over thecross-sectional width of the conveyor belt.

Electron beam emitter 492 (e.g., electron beam irradiation devicescommercially available from Titan Corporation, San Diego, Calif.) areconfigured to apply a 100 kilo-Gray dose of electrons applied at a powerof 300 kW. The electron beam emitters are scanning beam devices with asweep width of 1 meter to correspond to the width of the conveyor belt.In some embodiments, electron beam emitters with large, fixed beamwidths are used. Factors including belt/beam width, desired dose,biomass feedstock density, and power applied govern the number ofelectron beam emitters required for the plant to process 2,000 tons perday of dry feed.

Sonication

The irradiated biomass feedstock is mixed with water to form a slurrybefore ultrasonic energy is applied. There can be a separate sonicationsystem associated with each electron beam feed stream or severalelectron beam streams can be aggregated as feed for a single sonicationsystem.

In each sonication system, the irradiated biomass feedstock is fed intoa reservoir 1214 through a first intake 1232 and water is fed into thereservoir 1214 through second intake 1234. Appropriate valves (manual orautomated) control the flow of biomass feedstock and the flow of waterto produce a desired ratio of biomass feedstock to water (e.g., 10%cellulosic material, weight by volume). Each reservoir 1214 includes amixer 1240 to agitate the contents of volume 1236 and disperse biomassfeedstock throughout the water.

In each sonication system, the slurry is pumped (e.g., using a recessedimpeller vortex pump 1218) from reservoir 1214 to and through a flowcell 1224 including an ultrasonic transducer 1226. In some embodiments,pump 1218 is configured to agitate the slurry 1216 such that the mixtureof biomass feedstock and water is substantially uniform at inlet 1220 ofthe flow cell 1224. For example, the pump 1218 can agitate the slurry1216 to create a turbulent flow that persists throughout the pipingbetween the first pump and inlet 1220 of flow cell 1224.

Within the flow cell 1224, ultrasonic transducer 1226 transmitsultrasonic energy into slurry 1216 as the slurry flows through flow cell1224. Ultrasonic transducer 1226 converts electrical energy into highfrequency mechanical energy (e.g., ultrasonic energy) which is thendelivered to the slurry through booster 48. Ultrasonic transducers arecommercially available (e.g., from Hielscher USA, Inc. of Ringwood,N.J.) that are capable of delivering a continuous power of 16 kilowatts.

The ultrasonic energy traveling through booster 1248 in reactor volume1244 creates a series of compressions and rarefactions in process stream1216 with an intensity sufficient to create cavitation in process stream1216. Cavitation disaggregates components of the biomass feedstockincluding, for example, cellulosic and lignocellulosic materialdispersed in process stream 1216 (e.g., slurry). Cavitation alsoproduces free radicals in the water of process stream 1216 (e.g.,slurry). These free radicals act to further break down the cellulosicmaterial in process stream 1216. In general, about 250 MJ/m³ ofultrasonic energy is applied to process stream 1216 containing fragmentsof poplar chips. Other levels of ultrasonic energy (between about 5 andabout 4000 MJ/m³, e.g., 10, 25, 50, 100, 250, 500, 750, 1000, 2000, or3000) can be applied to other biomass feedstocks After exposure toultrasonic energy in reactor volume 1244, process stream 1216 exits flowcell 24 through outlet 1222.

Flow cell 1224 also includes a heat exchanger 1246 in thermalcommunication with at least a portion of reactor volume 1244. Coolingfluid 1248 (e.g., water) flows into heat exchanger 1246 and absorbs heatgenerated when process stream 1216 (e.g., slurry) is sonicated inreactor volume 1244. In some embodiments, the flow of cooling fluid 1248into heat exchanger 1246 is controlled to maintain an approximatelyconstant temperature in reactor volume 1244. In addition or in thealternative, the temperature of cooling fluid 1248 flowing into heatexchanger 1246 is controlled to maintain an approximately constanttemperature in reactor volume 1244.

The outlet 1242 of flow cell 1224 is arranged near the bottom ofreservoir 1214 to induce a gravity feed of process stream 1216 (e.g.,slurry) out of reservoir 1214 towards the inlet of a second pump 1230which pumps process stream 1216 (e.g., slurry) towards the primaryprocess subsystem.

Sonication systems can include a single flow path (as described above)or multiple parallel flow paths each with an associated individualsonication units. Multiple sonication units can also be arranged toseries to increase the amount of sonic energy applied to the slurry.

Primary Processes

A vacuum rotary drum type filter removes solids from the slurry beforefermentation. Liquid from the filter is pumped cooled prior to enteringthe fermentors. Filtered solids are passed to passed to thepost-processing subsystem for further processing.

The fermentation tanks are large, low pressure, stainless steel vesselswith conical bottoms and slow speed agitators. Multiple first stagefermentation tanks can be arranged in series. The temperature in thefirst stage fermentation tanks is controlled to 30 degrees centigradeusing external heat exchangers. Yeast is added to the first stagefermentation tank at the head of each series of tanks and carriesthrough to the other tanks in the series.

Second stage fermentation consists of two continuous fermentors inseries. Both fermentors are continuously agitated with slow speedmechanical mixers. Temperature is controlled with chilled water inexternal exchangers with continuous recirculation. Recirculation pumpsare of the progressive cavity type because of the high solidsconcentration.

Off gas from the fermentation tanks and fermentors is combined andwashed in a counter-current water column before being vented to theatmosphere. The off gas is washed to recover ethanol rather than for airemissions control.

Post-Processing Distillation

Distillation and molecular sieve adsorption are used to recover ethanolfrom the raw fermentation beer and produce 99.5% ethanol. Distillationis accomplished in two columns—the first, called the beer column,removes the dissolved CO₂ and most of the water, and the secondconcentrates the ethanol to a near azeotropic composition.

All the water from the nearly azeotropic mixture is removed by vaporphase molecular sieve adsorption. Regeneration of the adsorption columnsrequires that an ethanol water mixture be recycled to distillation forrecovery.

Fermentation vents (containing mostly CO₂, but also some ethanol) aswell as the beer column vent are scrubbed in a water scrubber,recovering nearly all of the ethanol. The scrubber effluent is fed tothe first distillation column along with the fermentation beer.

The bottoms from the first distillation contain all the unconvertedinsoluble and dissolved solids. The insoluble solids are dewatered by apressure filter and sent to a combustor. The liquid from the pressurefilter that is not recycled is concentrated in a multiple effectevaporator using waste heat from the distillation. The concentratedsyrup from the evaporator is mixed with the solids being sent to thecombustor, and the evaporated condensate is used as relatively cleanrecycle water to the process.

Because the amount of stillage water that can be recycled is limited, anevaporator is included in the process. The total amount of the waterfrom the pressure filter that is directly recycled is set at 25%.Organic salts like ammonium acetate or lactate, steep liquor componentsnot utilized by the organism, or inorganic compounds in the biomass endup in this stream. Recycling too much of this material can result inlevels of ionic strength and osmotic pressures that can be detrimentalto the fermenting organism's efficiency. For the water that is notrecycled, the evaporator concentrates the dissolved solids into a syrupthat can be sent to the combustor, minimizing the load to wastewatertreatment.

Wastewater Treatment

The wastewater treatment section treats process water for reuse toreduce plant makeup water requirements. Wastewater is initially screenedto remove large particles, which are collected in a hopper and sent to alandfill. Screening is followed by anaerobic digestion and aerobicdigestion to digest organic matter in the stream. Anaerobic digestionproduces a biogas stream that is rich in methane that is fed to thecombustor. Aerobic digestion produces a relatively clean water streamfor reuse in the process as well as a sludge that is primarily composedof cell mass. The sludge is also burned in the combustor. Thisscreening/anaerobic digestion/aerobic digestion scheme is standardwithin the current ethanol industry and facilities in the 1-5 milliongallons per day range can be obtained as “off-the-shelf” units fromvendors.

Combustor, Boiler, and Turbo-Generator

The purpose of the combustor, boiler, and turbo-generator subsystem isto burn various by-product streams for steam and electricity generation.For example, some lignin, cellulose, and hemicellulose remainsunconverted through the pretreatment and primary processes. The majorityof wastewater from the process is concentrated to a syrup high insoluble solids. Anaerobic digestion of the remaining wastewater producesa biogas high in methane. Aerobic digestion produces a small amount ofwaste biomass (sludge). Burning these by-product streams to generatesteam and electricity allows the plant to be self sufficient in energy,reduces solid waste disposal costs, and generates additional revenuethrough sales of excess electricity.

Three primary fuel streams (post-distillate solids, biogas, andevaporator syrup) are fed to a circulating fluidized bed combustor. Thesmall amount of waste biomass (sludge) from wastewater treatment is alsosent to the combustor. A fan moves air into the combustion chamber.Treated water enters the heat exchanger circuit in the combustor and isevaporated and superheated to 510° C. (950° F.) and 86 atm (1265 psia)steam. Flue gas from the combustor preheats the entering combustion airthen enters a baghouse to remove particulates, which are landfilled. Thegas is exhausted through a stack.

A multistage turbine and generator are used to generate electricity.Steam is extracted from the turbine at three different conditions forinjection into the pretreatment reactor and heat exchange indistillation and evaporation. The remaining steam is condensed withcooling water and returned to the boiler feedwater system along withcondensate from the various heat exchangers in the process. Treated wellwater is used as makeup to replace steam used in direct injection.

Other Embodiments

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.

In some embodiments, relatively low doses of radiation, optionally,combined with acoustic energy, e.g., ultrasound, are utilized tocrosslink, graft, or otherwise increase the molecular weight of anatural or synthetic carbohydrate-containing material, such as any ofthose materials in any form (e.g., fibrous form) described herein, e.g.,sheared or un-sheared cellulosic or lignocellulosic materials, such ascellulose. The cross-linking, grafting, or otherwise increasing themolecular weight of the natural or synthetic carbohydrate-containingmaterial can be performed in a controlled and predetermined manner byselecting the type or types of radiation employed (e.g., e-beam andultraviolet or e-beam and gamma) and/or dose or number of doses ofradiation applied. Such a material having increased molecular weight canbe useful in making a composite, such as a fiber-resin composite, havingimproved mechanical properties, such as abrasion resistance, compressionstrength, fracture resistance, impact strength, bending strength,tensile modulus, flexural modulus and elongation at break.Cross-linking, grafting, or otherwise increasing the molecular weight ofa selected material can improve the thermal stability of the materialrelative to an un-treated material. Increasing the thermal stability ofthe selected material can allow it to be processed at highertemperatures without degradation. In addition, treating materials withradiation can sterilize the materials, which can reduce their tendencyto rot, e.g., while in a composite. The cross-linking, grafting, orotherwise increasing the molecular weight of a natural or syntheticcarbohydrate-containing material can be performed in a controlled andpredetermined manner for a particular application to provide optimalproperties, such as strength, by selecting the type or types ofradiation employed and/or dose or doses of radiation applied.

When used, the combination of radiation, e.g., low dose radiation, andacoustic energy, e.g., sonic or ultrasonic energy, can improve materialthroughput and/or minimize energy usage.

The resin can be any thermoplastic, thermoset, elastomer, adhesive, ormixtures of these resins. Suitable resins include any resin, or mixtureof resins described herein.

In addition to the resin alone, the material having the increasedmolecular weight can be combined, blended, or added to other materials,such as metals, metal alloys, ceramics (e.g., cement), various inorganicand organic additives, such as lignin, elastomers, asphalts, glass, andmixtures of any of these and/or resins. When added to cement,fiber-reinforced cements can be produced having improved mechanicalproperties, such as the properties described herein, e.g., compressionstrength and/or fracture resistance.

Cross-linking, grafting, or otherwise increasing the molecular weight ofa natural or synthetic carbohydrate-containing material utilizingradiation can provide useful materials in many forms and for manyapplications. For example, the carbohydrate-containing material can bein the form of a paper product, such as paper, paper pulp, or papereffluent, particle board, glued lumber laminates, e.g., veneer, orplywood, lumber, e.g., pine, poplar, oak, or even balsa wood lumber.Treating paper, particle board, laminates or lumber, can increase theirmechanical properties, such as their strength. For example, treatingpine lumber with radiation can make a high strength structural material.

When paper is made using radiation, radiation can be utilized at anypoint in its manufacture. For example, the pulp can be irradiated, apressed fiber preform can be irradiated, or the finished paper itselfcan be irradiated. In some embodiments, radiation is applied at morethan one point during the manufacturing process.

For example, a fibrous material that includes a first cellulosic and/orlignocellulosic material having a first molecular weight can beirradiated in a manner to provide a second cellulosic and/orlignocellulosic material having a second molecular weight higher thanthe first molecular weight. For example, if gamma radiation is utilizedas the radiation source, a dose of from about 0.2 Mrad to about 10 Mrad,e.g., from about 0.5 Mrad to about 7.5 Mrad, or from about 2.0 Mrad toabout 5.0 Mrad, can be applied. If e-beam radiation is utilized, asmaller dose can be utilized (relative to gamma radiation), such as adose of from about 0.1 Mrad to about 5 Mrad, e.g., between about 0.2Mrad to about 3 Mrad, or between about 0.25 Mrad and about 2.5 Mrad.After the relatively low dose of radiation, the second cellulosic and/orlignocellulosic material can be combined with a material, such as aresin, and formed into a composite, e.g., by compression molding,injection molding or extrusion. Forming resin-fiber composites isdescribed in WO 2006/102543. Once composites are formed, they can beirradiated to further increase the molecular weight of thecarbohydrate-containing material while in the composite.

Alternatively, a fibrous material that includes a first cellulosicand/or lignocellulosic material having a first molecular weight can becombined with a material, such as a resin, to provide a composite, andthen the composite can be irradiated with a relatively low dose ofradiation so as to provide a second cellulosic and/or lignocellulosicmaterial having a second molecular weight higher than the firstmolecular weight. For example, if gamma radiation is utilized as theradiation source, a dose of from about 1 Mrad to about 10 Mrad can beapplied. Using this approach increases the molecular weight of thematerial while it is with a matrix, such as a resin matrix. In someembodiments, the resin is a cross-linkable resin, and, as such, itcross-links as the carbohydrate-containing material increases inmolecular weight, which can provide a synergistic effect to providemaximum mechanical properties to a composite. For example, suchcomposites can have excellent low temperature performance, e.g., havinga reduced tendency to break and/or crack at low temperatures, e.g.,temperatures below 0° C., e.g., below −10° C., −20° C., −40° C., −50°C., −60° C. or even below −100° C., and/or excellent performance at hightemperatures, e.g., capable of maintaining their advantageous mechanicalproperties at relatively high temperature, e.g., at temperatures above100° C., e.g., above 125° C., 150° C., 200° C., 250° C., 300° C., 400°C., or even above 500° C. In addition, such composites can haveexcellent chemical resistance, e.g., resistance to swelling in asolvent, e.g., a hydrocarbon solvent, resistance to chemical attack,e.g., by strong acids, strong bases, strong oxidants (e.g., chlorine orbleach) or reducing agents (e.g., active metals such as sodium andpotassium).

In some embodiments, the resin, or other matrix material, does notcrosslink during irradiation. In some embodiments, additional radiationis applied while the carbohydrate-containing material is within thematrix to further increase the molecular weight of thecarbohydrate-containing material. In some embodiments, the radiationcauses bonds to form between the matrix and the carbohydrate-containingmaterial.

In some embodiments, the carbohydrate-containing material is in the formof fibers. In such embodiments, when the fibers are utilized in acomposite, the fibers can be randomly oriented within the matrix. Inother embodiments, the fibers can be substantially oriented, such as inone, two, three or four directions. If desired, the fibers can becontinuous or discrete.

Any of the following additives can added to the fibrous materials,densified fibrous materials a or any other materials and compositesdescribed herein. Additives, e.g., in the form of a solid, a liquid or agas, can be added, e.g., to the combination of a fibrous material andresin. Additives include fillers such as calcium carbonate, graphite,wollastonite, mica, glass, fiber glass, silica, and talc; inorganicflame retardants such as alumina trihydrate or magnesium hydroxide;organic flame retardants such as chlorinated or brominated organiccompounds; ground construction waste; ground tire rubber; carbon fibers;or metal fibers or powders (e.g., aluminum, stainless steel). Theseadditives can reinforce, extend, or change electrical, mechanical orcompatibility properties. Other additives include lignin, fragrances,coupling agents, compatibilizers, e.g., maleated polypropylene,processing aids, lubricants, e.g., fluorinated polyethylene,plasticizers, antioxidants, opacifiers, heat stabilizers, colorants,foaming agents, impact modifiers, polymers, e.g., degradable polymers,photostabilizers, biocides, antistatic agents, e.g., stearates orethoxylated fatty acid amines. Suitable antistatic compounds includeconductive carbon blacks, carbon fibers, metal fillers, cationiccompounds, e.g., quaternary ammonium compounds, e.g.,N-(3-chloro-2-hydroxypropyl)-trimethylammonium chloride, alkanolamides,and amines. Representative degradable polymers include polyhydroxyacids, e.g., polylactides, polyglycolides and copolymers of lactic acidand glycolic acid, poly(hydroxybutyric acid), poly(hydroxyvaleric acid),poly[lactide-co-(e-caprolactone)], poly[glycolide-co-(e-caprolactone)],polycarbonates, poly(amino acids), poly(hydroxyalkanoate)s,polyanhydrides, polyorthoesters and blends of these polymers.

When described additives are included, they can be present in amounts,calculated on a dry weight basis, of from below 1 percent to as high as80 percent, based on total weight of the fibrous material. Moretypically, amounts range from between about 0.5 percent to about 50percent by weight, e.g., 5 percent, 10 percent, 20 percent, 30, percentor more, e.g., 40 percent.

Any additives described herein can be encapsulated, e.g., spray dried ormicroencapsulated, e.g., to protect the additives from heat or moistureduring handling.

The fibrous materials, densified fibrous materials, resins or additivesmay be dyed. For example, the fibrous material can be dyed beforecombining with the resin and compounding to form composites. In someembodiments, this dyeing can be helpful in masking or hiding the fibrousmaterial, especially large agglomerations of the fibrous material, inmolded or extruded parts, when this is desired. Such largeagglomerations, when present in relatively high concentrations, can showup as speckles in the surfaces of the molded or extruded parts.

For example, the desired fibrous material can be dyed using an acid dye,direct dye or a reactive dye. Such dyes are available from Spectra Dyes,Kearny, N.J. or Keystone Aniline Corporation, Chicago, Ill. Specificexamples of dyes include SPECTRA™ LIGHT YELLOW 2G, SPECTRACID™ YELLOW4GL CONC 200, SPECTRANYL™ RHODAMINE 8, SPECTRANYL™ NEUTRAL RED B,SPECTRAMINE™ BENZOPERPURINE, SPECTRADIAZO™ BLACK OB, SPECTRAMINE™TURQUOISE G, and SPECTRAMINE™ GREY LVL 200%, each being available fromSpectra Dyes.

In some embodiments, resin color concentrates containing pigments areblended with dyes. When such blends are then compounded with the desiredfibrous material, the fibrous material may be dyed in-situ during thecompounding. Color concentrates are available from Clariant.

It can be advantageous to add a scent or fragrance to the fibrousmaterials, densified fibrous or composites. For example, it can beadvantageous for the composites smell and/or look like natural wood,e.g., cedar wood. For example, the fragrance, e.g., natural woodfragrance, can be compounded into the resin used to make the composite.In some implementations, the fragrance is compounded directly into theresin as an oil. For example, the oil can be compounded into the resinusing a roll mill, e.g., a BANBURY® mixer or an extruder, e.g., atwin-screw extruder with counter-rotating screws. An example of aBANBURY mixer is the F-Series BANBURY mixer, manufactured by Farrel. Anexample of a twin-screw extruder is the WP ZSK 50 MEGACOMPOUNDER™,manufactured by Krupp Werner & Pfleiderer. After compounding, thescented resin can be added to the fibrous material and extruded ormolded. Alternatively, master batches of fragrance-filled resins areavailable commercially from International Flavors and Fragrances, underthe tradename POLYIFF™ or from the RTP Company. In some embodiments, theamount of fragrance in the composite is between about 0.005% by weightand about 10% by weight, e.g., between about 0.1% and about 5% or 0.25%and about 2.5%.

Other natural wood fragrances include evergreen or redwood. Otherfragrances include peppermint, cherry, strawberry, peach, lime,spearmint, cinnamon, anise, basil, bergamot, black pepper, camphor,chamomile, citronella, eucalyptus, pine, fir, geranium, ginger,grapefruit, jasmine, juniperberry, lavender, lemon, mandarin, marjoram,musk, myrhh, orange, patchouli, rose, rosemary, sage, sandalwood, teatree, thyme, wintergreen, ylang ylang, vanilla, new car or mixtures ofthese fragrances. In some embodiments, the amount of fragrance in thefibrous material-fragrance combination is between about 0.005% by weightand about 20% by weight, e.g., between about 0.1% and about 5% or 0.25%and about 2.5%.

While fibrous materials have been described, such as cellulosic andlignocellulosic fibrous materials, other fillers may be used for makingthe composites. For example, inorganic fillers such as calcium carbonate(e.g., precipitated calcium carbonate or natural calcium carbonate),aragonite clay, orthorhombic clays, calcite clay, rhombohedral clays,kaolin, clay, bentonite clay, dicalcium phosphate, tricalcium phosphate,calcium pyrophosphate, insoluble sodium metaphosphate, precipitatedcalcium carbonate, magnesium orthophosphate, trimagnesium phosphate,hydroxyapatites, synthetic apatites, alumina, silica xerogel, metalaluminosilicate complexes, sodium aluminum silicates, zirconiumsilicate, silicon dioxide or combinations of the inorganic additives maybe used. The fillers can have, e.g., a particle size of greater than 1micron, e.g., greater than 2 micron, 5 micron, 10 micron, 25 micron oreven greater than 35 microns.

Nanometer scale fillers can also be used alone, or in combination withfibrous materials of any size and/or shape. The fillers can be in theform of, e.g., a particle, a plate, or a fiber. For example, nanometersized clays, silicon and carbon nanotubes, and silicon and carbonnanowires can be used. The filler can have a transverse dimension lessthan 1000 nm, e.g., less than 900 nm, 800 nm, 750 nm, 600 nm, 500 nm,350 nm, 300 nm, 250 nm, 200 nm, less than 100 nm, or even less than 50nm.

In some embodiments, the nano-clay is a montmorillonite. Such clays areavailable from Nanocor, Inc. and Southern Clay products, and have beendescribed in U.S. Pat. Nos. 6,849,680 and 6,737,464. The clays can besurface treated before mixing into, e.g., a resin or a fibrous material.For example, the clay can be surface is treated so that its surface isionic in nature, e.g., cationic or anionic.

Aggregated or agglomerated nanometer scale fillers, or nanometer scalefillers that are assembled into supramolecular structures, e.g.,self-assembled supramolecular structures can also be used. Theaggregated or supramolecular fillers can be open or closed in structure,and can have a variety of shapes, e.g., cage, tube or spherical.

Mobile Biomass Processing

Stationary processing facilities for processing biomass have beendescribed. However, depending upon the source of biomass feedstock andthe products produced therefrom, it can be advantageous to process orpartially process biomass in mobile facilities that can be located closeto the source of the feedstock and/or close to target markets forproducts produced from the feedstock. As an example, in someembodiments, various grasses such as switchgrass are used as biomassfeedstock. Transporting large volumes of switchgrass from fields whereit grows to processing facilities hundreds or even thousands of milesaway may be both wasteful energetically and economically costly (forexample, transportation of feedstock by train is estimated to costbetween $3.00 and $6.00 per ton per 500 miles). Moreover, some of theproducts of processing switchgrass feedstock may be suitable for marketsin regions where biomass feedstock is grown (e.g., ruminant feed forlivestock). Once again, transporting ruminant feed hundreds or thousandsof miles to market may not be economically viable.

Accordingly, in some embodiments, the processing systems disclosedherein are implemented as mobile, reconfigurable processing facilities.One embodiment of such a mobile facility is shown in FIG. 63. Processingfacility 8000 includes five transport trucks 8002, 8004, 8006, 8008, and8010 (although five trucks are shown in FIG. 63, in general, any numberof trucks may be used). Truck 8002 includes water supply and processingsystems and electrical supply systems for the other trucks. Trucks 8004,8006, 8008, and 8010 are each configured to process biomass feedstock inparallel.

Truck 8002 includes a water supply inlet 8012 for receiving water from acontinuous supply (such as a water main) or a reservoir (e.g., a tank onanother truck, or a tank or other reservoir located at the processingsite). Process water is circulated to each of trucks 8004, 8006, 8008,and 8010 through a water supply conduit 8020. Each of trucks 8004, 8006,8008, and 8010 includes a portion of conduit 8020. When the trucks arepositioned next to one another to set up the mobile processing facility,the portions of conduit 8020 are connected to form a continuous watertransport conduit. Each of trucks 8004, 8006, 8008, and 8010 includes awater inlet 8022 to supply process water, and a water outlet 8024 toremove used process water. The water outlets 8024 in each of trucks8004, 8006, 8008, and 8010 lead to a piecewise continuous water disposalconduit 8026, which is similarly joined into a continuous conduit whenthe trucks are positioned next to one another. Waste process water iscirculated to water processor 8028 in truck 8002, which treats the waterto remove harmful waste materials and then recycles the treated watervia conduit 8030 back into supply conduit 8020. Waste materials removedfrom the used process water can be disposed of on site, or stored (e.g.,in another truck, not shown) and transported to a storage facility.

Truck 8002 also includes an electrical supply station 8016 that provideselectrical power to each of trucks 8004, 8006, 8008, and 8010.Electrical supply station 8016 can be connected to an external powersource via connection 8014. Alternatively, or in addition, electricalsupply station can be configured to generate power (e.g., via combustionof a fuel source). Electrical power is supplied to each of trucks 8004,8006, 8008, and 8010 via electrical supply conduit 8040. Each of trucks8004, 8006, 8008, and 8010 includes an electrical power terminal 8018 towhich devices on the truck requiring electrical power are connected.

Each of trucks 8004, 8006, 8008, and 8010 includes a feedstock inlet8042 and a waste outlet 8044. Biomass feedstock enters each of trucks8004, 8006, 8008, and 8010 through inlet 8042, where it is processedaccording to the methods disclosed herein. Following processing, wastematerial is discharged through outlet 8044. Alternatively, in someembodiments, each of trucks 8004, 8006, 8008, and 8010 can be connectedto a common feedstock inlet (e.g., positioned in truck 8002), and eachtruck can discharge waste material through a common outlet (e.g., alsopositioned in truck 8002).

Each of trucks 8004, 8006, 8008, and 8010 can include various types ofprocessing units; for example, in the configuration shown in FIG. 63,each of trucks 8004, 8006, 8008, and 8010 includes an ion accelerator8032 (e.g., a horizontal Pelletron-based tandem folded accelerator), aheater/pyrolysis station 8034, a wet chemical processing unit 8036, anda biological processing unit 8038. In general, each of trucks 8004,8006, 8008, and 8010 can include any of the processing systems disclosedherein. In certain embodiments, each of trucks 8004, 8006, 8008, and8010 will include the same processing systems. In some embodiments,however, one or more trucks may have different processing systems.

In addition, some or all trucks may have certain processing systemsonboard, but which are not used, depending upon the nature of thefeedstock. In general, the layout of the various onboard processingsystems on each of trucks 8004, 8006, 8008, and 8010 is reconfigurableaccording to the type of material that is processed.

Processing facility 8000 is an exemplary parallel processing facility;each of trucks 8004, 8006, 8008, and 8010 processes biomass feedstock inparallel. In certain embodiments, mobile processing facilities areimplemented as serial processing facilities. An embodiment oftrain-based serial mobile processing facility 8500 is shown in FIG. 64.Processing facility 8500 includes three rail cars 8502, 8504, and 8506(in general, any number of rail cars can be used), each configured toperform one or more processing steps in an overall biomass processingprocedure. Rail car 8502 includes a feedstock inlet for receivingfeedstock from a storage repository (e.g., a storage building, oranother rail car). Feedstock is conveyed from one processing unit toanother among the three rail cars via a continuous conveyor system. Railcar 8502 also includes an electrical supply station 8514 for supplyingelectrical power to each of rail cars 8502, 8504, and 8506.

Rail car 8502 includes a coarse mechanical processor 8516 and a finemechanical processor 8518 for converting raw feedstock to a finelydivided fibrous material. A third mechanical processor 8520 rolls thefibrous material into a flat, continuous mat. The mat of fibrousmaterial is then transported to an ion accelerator 8522 on rail car 8504that exposes the fibrous material to an ion beam. Following exposure tothe ion beam, the fibrous material is transported to a low energyelectron accelerator 8524.

The fibrous material is subsequently transported to a chemicalprocessing unit 8526 on rail car 8506 for one or more chemical treatmentsteps. Rail car 8506 includes a process water inlet 8532 which receivesprocess water from an external reservoir (e.g., a tank or another railcar).

Following chemical treatment in processing unit 8526, the material istransported to a biological processing unit 8528 to initiatefermentation of liberated sugars from the material. After biologicalprocessing is complete, the material is transported to a separator 8530,which diverts useful products into conduit 8510 and waste materials intoconduit 8512. Conduit 8510 can be connected to a storage unit (e.g., atanker car or an external storage tank). Similarly, waste products canbe conveyed through conduit 8512 to a storage unit such as a tanker car,and/or to an external storage facility. Separator 8530 also recyclesclean process water for subsequent delivery to chemical processing unit8536 and/or biological processing unit 8528.

As discussed previously, processing facility 8500 is an example of asequential configuration of a mobile processing facility; each of railcars 8502, 8504, and 8506 includes a different subset of processingsystems; and the feedstock process flow from each car is connected tothe next car in series to complete the processing sequence.

In general, a wide variety of different mobile processing configurationscan be used to process biomass feedstock. Both truck-based andtrain-based mobile processing facilities can be configured for eitherserial operation or parallel operation. Generally, the layout of thevarious processing units is reconfigurable, and not all processing unitscan be used for particular feedstocks. When a particular processing unitis not used for a certain feedstock, the processing unit can bewithdrawn from the process flow. Alternatively, the processing unit canremain in the overall process flow, but can be deactivated so thatfeedstock passes through the deactivated unit rapidly without beingmodified.

Mobile processing facilities can include one or more electronic controldevices that automate some or all aspects of the biomass processingprocedure and/or the mobile facility setup procedure. For example, anelectronic control device can be configured to receive input informationabout a feedstock material that is to be processed, and can generate avariety of output information including a suggested configuration of themobile processing facility, and/or values for one or more processparameters involved in the biomass processing procedure that will beimplemented.

Treatment of Hydrocarbon-Containing Materials

In some embodiments, the methods and systems disclosed herein fortreating biomass can be used to process hydrocarbon-containing materialssuch as tar or oil sands, oil shale, crude oil (e.g., heavy crude oiland/or light crude oil), bitumen, coal, petroleum gases (e.g., methane,ethane, propane, butane, isobutane), liquefied natural and/or syntheticgas, asphalt, and other natural materials that include various types ofhydrocarbons. For example, a processing facility forhydrocarbon-containing materials receives a supply of material. Thematerial can be delivered directly from a mine, e.g., by conveyor beltand/or rail car system, and in certain embodiments, the processingfacility can be constructed in relatively close proximity to, or evenatop, the mine. In some embodiments, the material can be transported tothe processing facility via railway freight car or another motorizedtransport system, and/or pumped to the processing facility via pipeline.

When the material enters the processing facility, the material can bebroken down mechanically and/or chemically to yield starting material.As an example, the material can include material derived from oil sandsand containing crude bitumen. Bitumen can then be processed into one ormore hydrocarbon products using the methods disclosed herein. In someembodiments, the oil sands material can be extracted from surface minessuch as open pit mines. In certain embodiments, sub-surface oil sandsmaterial can be extracted using a hot water flotation process thatremoves oil from sand particles, and then adding naphtha to allowpumping of the oil to the processing facility.

Bitumen processing generally includes two stages. In a first stage,relatively large bitumen hydrocarbons are cracked into smaller moleculesusing coking, hydrocracking, or a combination of the two techniques. Inthe coking process, carbon is removed from bitumen hydrocarbon moleculesat high temperatures (e.g., 400° C. or more), leading to cracking of themolecules. In hydrocracking, hydrogen is added to bitumen molecules,which are then cracked over a catalyst system (e.g., platinum).

In a second stage, the cracked bitumen molecules are hydrotreated. Ingeneral, hydrotreating includes heating the cracked bitumen molecules ina hydrogen atmosphere to remove metals, nitrogen (e.g., as ammonia), andsulfur (e.g., as elemental sulfur).

The overall bitumen processing procedure typically producesapproximately one barrel of synthetic crude oil for every 2.5 tons ofoil sand material processed. Moreover, an energy equivalent ofapproximately one barrel of oil is used to produce three barrels ofsynthetic crude oil from oil sand-derived bitumen sources.

As another example, oil shale typically includes fine-grainedsedimentary rock that includes significant amounts of kerogen (a mixtureof various organic compounds in solid form). By heating oil shale, avapor is liberated which can be purified to yield a hydrocarbon richshale oil and a combustible hydrocarbon shale gas. Typically, the oilshale is heated to between 250° C. and 550° C. in the absence of oxygento liberate the vapor.

The efficiency and cost-effectiveness with which usable hydrocarbonproducts can be extracted from oil sands material, oil shale, crude oil,and other oil-based materials can be improved by applying the methodsdisclosed herein. In addition, a variety of different hydrocarbonproducts (including various hydrocarbon fractions that are present inthe material, and other types of hydrocarbons that are formed duringprocessing) can be extracted from the materials.

In some embodiments, for example, ion beams can be used to processmaterials (and/or intermediate materials derived from the materials).For example, ion beams that include one or more different types of ions(e.g., protons, carbon ions, oxygen ions, hydride ions) can be used toprocess materials. The ion beams can include positive ions and/ornegative ions, in doses that vary from 1 Mrad to 2500 Mrad or more,e.g., 50, 100, 250, 350, 500, 1000, 1500, 2000, or 2500 Mrad, or evenhigher levels.

In some embodiments, metal ions can be used to treat biomass material inaddition to, or as alternatives to, the various types of ions disclosedabove. For example, ions of metals that function as catalysts inhydrocarbon cracking, reforming, and alkylation processes, such as ionsof rhodium, iridium, and platinum, can be generated, and biomassmaterials can be exposed to such ions to initiate degradation reactionsin the biomass. Processing steps such as hydrocarbon cracking can alsobe used before, during, and/or after exposure to metal ions.

In certain embodiments, other methods can also be used to process rawand/or intermediate materials. For example, raw and/or intermediatematerials can be exposed to electron beams. In general, the electronbeams can have any of the properties discussed previously with regard tobiomass processing. In some embodiments, additional processing methodscan be used, including oxidation, pyrolysis, and sonication. In general,process parameters for each of these techniques when treatinghydrocarbon-based raw and/or intermediate materials can be the same asthose disclosed above in connection with biomass materials. Variouscombinations of these techniques can also be used to process raw orintermediate materials.

Generally, the various techniques can be used in any order, and anynumber of times, to treat raw and/or intermediate materials. Forexample, to process bitumen from oil sands, one or more of thetechniques disclosed herein can be used prior to any mechanicalbreakdown steps, following one or more mechanical breakdown steps, priorto cracking, after cracking and/or prior to hydrotreatment, and afterhydrotreatment. As another example, to process oil shale, one or more ofthe techniques disclosed herein can be used prior to either or both ofthe vaporization and purification steps discussed above. Productsderived from the hydrocarbon-based materials can be treated again withany combination of techniques prior to transporting the products out ofthe processing facility (e.g., either via motorized transport, or viapipeline).

The techniques disclosed herein can be applied to process raw and/orintermediate material in dry form, in a solution or slurry, or ingaseous form (e.g., to process hydrocarbon vapors at elevatedtemperature). The solubility of raw or intermediate products insolutions and slurries can be controlled through selective addition ofone or more agents such as acids, bases, oxidizing agents, reducingagents, and salts. In general, the methods disclosed herein can be usedto initiate and/or sustain the reaction of raw and/or intermediatehydrocarbon-containing materials, extraction of intermediate materialsfrom materials (e.g., extraction of hydrocarbon components from othersolid or liquid components), distribution of raw and/or intermediatematerials, and separation of intermediate materials from materials(e.g., separation of hydrocarbon-containing components from other solidmatrix components to increase the concentration and/or purity and/orhomogeneity of the hydrocarbon components).

In addition, microorganisms can be used for processing raw orintermediate materials, either prior to or following the use of ion beamexposure, electron beam exposure, pyrolysis, oxidation, sonication,and/or chemical processing. Suitable microorganisms include varioustypes of bacteria, yeasts, and mixtures thereof, as disclosedpreviously. The processing facility can be equipped to remove harmfulbyproducts that result from the processing of raw or intermediatematerials, including gaseous products that are harmful to humanoperators, and chemical byproducts that are harmful to humans and/orvarious microorganisms.

In some embodiments, the use of one or more of the techniques disclosedherein results in a molecular weight reduction of one or more componentsof the raw or intermediate material that is processed. As a result,various lower weight hydrocarbon substances can be produced from one ormore higher weight hydrocarbon substances. In certain embodiments, theuse of one or more of the techniques disclosed herein results in anincrease in molecular weight of one or more components of the raw orintermediate material that is processed. For example, the varioustechniques disclosed herein can induce bond-formation between moleculesof the components, leading to the formation of increased quantities ofcertain products, and even to new, larger weight products. In additionto hydrocarbon products, various other compounds can be extracted fromthe materials, including nitrogen based compounds (e.g., ammonia),sulfur-based compounds, and silicates and other silicon-based compounds.In certain embodiments, one or more products extracted from thematerials can be combusted to generate process heat for heating water,raw or intermediate materials, generating electrical power, or for otherapplications.

In some embodiments, processing raw and/or intermediate materials viaone or more of ion beam exposure, electron beam exposure, pyrolysis,oxidation, sonication, microorganisms, and/or chemical processing canlead to improvements in the efficiency (and even the elimination) ofother processing steps. For example, processing oil sand materials(including bitumen) using one or more of the techniques disclosed hereincan lead to more efficient cracking and/or hydrotreatment of thebitumen. As another example, processing oil shale can lead to moreefficient extraction of various products, including shale oil and/orshale gas, from the oil shale. In certain embodiments, steps such ascracking or vaporization may not even be necessary if the techniquesdisclosed herein are first used to treat the material. Further, in someembodiments, by treating raw and/or intermediate materials, the productscan be made more soluble in certain solvents, in preparation forsubsequent processing steps in solution (e.g., steam blasting,sonication). Improving the solubility of the products can improve theefficiency of subsequent solution-based treatment steps. By improvingthe efficiency of other processing steps (e.g., cracking and/orhydrotreatment of bitumen, vaporization of oil shale), the overallenergy consumed in processing the materials can be reduced, makingextraction and processing of the materials economically feasible.

In certain embodiments, ion beams can be particularly efficient atprocessing raw hydrocarbon-containing materials. For example, due to theability of ion beams to initiate both polymerization anddepolymerization reactions, to deposit heat in the irradiated material,and to sputter or otherwise displace atoms of the irradiated material,hydrocarbon materials such as oil sands, oil shale, crude oil, asphalt,and other materials can be treated to improve additional processingsteps for these materials and/or to extract useful products from thematerials.

Products derived from processing hydrocarbon-containing materials caninclude one or more compounds suitable for use as fuels. The fuelcompounds can be used on-site (e.g., combusted to generate electricalpower) and/or can be transported to another facility for storage and/oruse.

Processing of Crude Oil

The methods and systems disclosed herein can be used to process crudeoil in addition to, or as an alternative to, conventional oil refiningtechnologies. In particular, ion beam treatment methods—alone or incombination with any of the other methods disclosed herein—can be usedfor low temperature oil cracking, reforming, functionalization, andother processes.

Crude oils typically include large numbers of different hydrocarbonspecies, ranging from relatively light, volatile, low molecular weighthydrocarbons, to heavy, dense, highly viscous fractions (e.g., heavyoil, bitumen) of high molecular weight. The heavy crudes typicallycontain more sulfur and/or nitrogen and/or metals, relative to lighter,sweeter crudes such as the West Texas Intermediate which is traded onthe New York Mercantile Exchange. In general, sweet crudes includerelatively low amounts of sulfur-containing compounds; the sour crudesinclude larger amounts of sulfur-containing compounds. Simple refineriesare generally designed to handle sweet crudes, while more complex deepconversion refineries are required for the processing of heavy, sourcrude oils.

The large number of different hydrocarbon (and other) species in crudeoil typically establish a relatively delicately balanced colloidalsolubility system. When certain properties of the crude oil are changed(e.g., temperature, pressure, composition), the solubility balance canbe destabilized, causing a single-phase crude oil feedstock to change toa multiphase, multicomponent mixture (which can include one or more gas,liquid, and solid phases). At room temperature and pressure, variouscomponents of crude oil are in different physical states. For example,lighter hydrocarbons (e.g., methane, ethane, propane, butane) are gasesat room temperature and pressure. Components of intermediate molecularweight (e.g., pentane, hexane, octane, gasoline, kerosene, diesel fuel)are liquids under these conditions. Heavy fractions (e.g., asphalt, wax)are solids at standard temperature and pressure. Due to this range ofphysical states, conventional refineries typically process crude oil atelevated temperatures and/or pressures to ensure that most (or all) ofthe hydrocarbon fractions in the crude are either liquids or gases

In some embodiments, one or more of the pretreatment methods disclosedherein, include ion beam pretreatment alone or in combination with oneor more of the other techniques disclosed herein, can be used to enableprocessing of crude oil at reduced temperature and/or pressure. Forexample, the crude oil can be exposed to an ion beam, which assists inbreaking molecular bonds in heavy crude oil fractions, producing lowermolecular weight products as a result. While the heavy fractions aretypically highly viscous liquids or even solids, the lower molecularweight products are typically less viscous liquids. As a result, theproducts can be further processed and/or refined at lower temperatureand/or pressure. In certain embodiments, for example, following ion beamexposure, the exposed crude oil can be processed at a temperature of800° F. or less (e.g., 700° F. or less, 600° F. or less, 500° F. orless, 400° F. or less, 300° F. or less, 200° F. or less, 150° F. orless, 100° F. or less, 50° F. or less).

In some embodiments, following ion beam exposure, the exposed crude oilcan be processed at a pressure of 100 atmospheres or less (e.g., 90atmospheres or less, 80 atmospheres or less, 70 atmospheres or less, 60atmospheres or less, 50 atmospheres or less, 40 atmospheres or less, 30atmospheres or less, 20 atmospheres or less, 10 atmospheres or less, 5atmospheres or less, 2 atmospheres or less).

Crude oil refining comprises processes that separate various hydrocarbonand other components in the oil and, in some cases, convert certainhydrocarbons to other hydrocarbon species via molecular rearrangement(e.g., chemical reactions that break bonds). In some embodiments, afirst step in the refining process is a water washing step to removesoluble components such as salts from the crude oil. Typically, thewashed crude oil is then directed to a furnace for preheating. Asdiscussed above, the crude oil can include a large number of differentcomponents with different viscosities; some components may even be solidat room temperature. By heating the crude oil, the component mixture canbe converted to a mixture that can be flowed from one processing systemto another (and from one end of a processing system to the other) duringrefining.

Preheated crude is then sent to a distillation tower, wherefractionation of various components in the crude oil mixture occurs withheating in a distillation column. The amount of heat energy supplied tothe crude oil mixture in the distillation process depends in part uponthe composition of the oil; in general, however, significant energy isexpended in heating the crude oil during distillation, cooling thedistillates, pressurizing the distillation column, and in other suchsteps. Within limits, certain refineries are capable of reconfigurationto handle differing crude oil feedstocks and products. In general,however, due to the relatively specialized refining apparatus, theability of refineries to handle significantly different crude oilfeedstocks is restricted.

In some embodiments, pretreatment of crude oil feedstocks using methodsdisclosed herein, such as ion beam pretreatment (and/or one or moreadditional pretreatments), can enhance the ability of a refiningapparatus to accept crude oils having different compositions. Forexample, by exposing a crude oil stream to incident ions from an ionbeam, various chemical and/or physical properties of the crude oilmixture can be changed. Incident ions can cause chemical bonds to break,leading to the production of lighter molecular weight components withlower viscosities from heavier components with higher viscosities.Alternatively, or in addition, exposure of certain components to ionscan lead to isomerization of the exposed components. The newly formedisomers can have lower viscosities than the components from which theyare formed. The lighter molecular weight components and/or isomers withlower viscosities can then be introduced into the refinery, enablingprocessing of crude oil feedstock while may not have been suitable forprocessing initially.

In general, the various components of crude oil distill at differenttemperature ranges, corresponding to different vertical heights in adistillation column. Typically, for example, a refinery distillationcolumn will include product streams at a large number of differenttemperature cut ranges, with the lowest boiling point (and, generally,smallest molecular weight) components drawn from the top of the column,and the highest boiling point, heaviest molecular weight componentsdrawn from lower levels of the column. As an example, light distillatesextracted from upper regions of the column typically include one or moreof aviation gasoline, motor gasoline, napthas, kerosene, and refinedoils. Intermediate distillates, removed from the middle region of thecolumn, can include one or more of gas oil, heavy furnace oil, anddiesel fuel oil. Heavy distillates, which are generally extracted fromlower levels of the column, can include one or more of lubricating oil,grease, heavy oils, wax, and cracking stock. Residues remaining in thestill can include a variety of high boiling components such aslubricating oil, fuel oil, petroleum jelly, road oils, asphalt, andpetroleum coke. Certain other products can also be extracted from thecolumn, including natural gas (which can be further refined and/orprocessed to produce components such as heating fuel, natural gasoline,liquefied petroleum gas, carbon black, and other petrochemicals), andvarious by-products (including, for example, fertilizers, ammonia, andsulfuric acid).

Generally, treatment of crude oil and/or components thereof using themethods disclosed herein (including, for example, ion beam treatment,alone or in combination with one or more other methods) can be used tomodify molecular weights, chemical structures, viscosities,solubilities, densities, vapor pressures, and other physical propertiesof the treated materials. Typical ions that can be used for treatment ofcrude oil and/or components thereof can include protons, carbon ions,oxygen ions, and any of the other types of ions disclosed herein. Inaddition, ions used to treat crude oil and/or its components can includemetal ions; in particular, ions of metals that catalyze certain refineryprocesses (e.g., catalytic cracking) can be used to treat crude oiland/or components thereof. Exemplary metal ions include, but are notlimited to, platinum ions, palladium ions, iridium ions, rhodium ions,ruthenium ions, aluminum ions, rhenium ions, tungsten ions, and osmiumions.

In some embodiments, multiple ion exposure steps can be used. A firstion exposure can be used to treat crude oil (or components thereof) toeffect a first change in one or more of molecular weight, chemicalstructure, viscosity, density, vapor pressure, solubility, and otherproperties. Then, one or more additional ion exposures can be used toeffect additional changes in properties. As an example, the first ionexposure can be used to convert a substantial fraction of one or morehigh boiling, heavy components to lower molecular weight compounds withlower boiling points. Then, one or more additional ion exposures can beused to cause precipitation of the remaining amounts of the heavycomponents from the component mixture.

In general, a large number of different processing protocols can beimplemented, according to the composition and physical properties of thefeedstock. In certain embodiments, the multiple ion exposures caninclude exposures to only one type of ion. In some embodiments, themultiple ion exposures can include exposures to more than one type ofion. The ions can have the same charges, or different charge magnitudesand/or signs.

In certain embodiments, the crude oil and/or components thereof can beflowed during exposure to ion beams. Exposure during flow can greatlyincrease the throughput of the exposure process, enablingstraightforward integration with other flow-based refinery processes.

In some embodiments, the crude oil and/or components thereof can befunctionalized during exposure to ion beams. For example, thecomposition of one or more ion beams can be selected to encourage theaddition of particular functional groups to certain components (or allcomponents) of a crude oil feedstock. One or more functionalizing agents(e.g., ammonia) can be added to the feedstock to introduce particularfunctional groups. By functionalizing the crude oil and/or componentsthereof, ionic mobility within the functionalized compounds can beincreased (leading to greater effective ionic penetration duringexposure), and physical properties such as viscosity, density, andsolubility of the crude oil and/or components thereof can be altered. Byaltering one or more physical properties of the crude oil and/or crudeoil components, the efficiency and selectivity of subsequent refiningsteps can be adjusted, and the available product streams can becontrolled. Moreover, functionalization of crude oil and/or crude oilcomponents can lead to improved activating efficiency of catalysts usedin subsequent refining steps.

In general, the methods disclosed herein—including ion beam exposure ofcrude oil and crude oil components—can be performed before, during, orafter any of the other refining steps disclosed herein, and/or before,during, or after any other steps that are used to refine crude oil. Themethods disclosed herein can also be used after refining is complete,and/or before refining begins. In certain embodiments, the methodsdisclosed herein, including ion beam exposure, can be used to processcrude oil even during extraction of the crude oil from oil fields.

In some embodiments, when crude oil and/or components thereof areexposed to one or more ion beams, the exposed material can also beexposed to one or more gases concurrent with ion beam exposure. Certaincomponents of crude oil, such as components that include aromatic rings,may be relatively more stable to ion beam exposure than non-aromaticcomponents. Typically, for example, ion beam exposure leads to theformation of reactive intermediates such as radicals from hydrocarbons.The hydrocarbons can then react with other less reactive hydrocarbons.To reduce the average molecular weight of the exposed material,reactions between the reactive products and less reactive hydrocarbonslead to molecular bond-breaking events, producing lower weight fragmentsfrom longer chain molecules. However, more stable reactive intermediates(e.g., aromatic hydrocarbon intermediates) may not react with otherhydrocarbons, and can even undergo polymerization, leading to theformation of heavier weight compounds. To reduce the extent ofpolymerization in ion beam exposed crude oil and/or crude oilcomponents, one or more radical quenchers can be introduced during ionbeam exposure. The radical quenchers can cap reactive intermediates,preventing the re-formation of chemical bonds that have been broken bythe incident ions. Suitable radical quenchers include hydrogen donorssuch as hydrogen gas.

In certain embodiments, reactive compounds can be introduced during ionbeam exposure to further promote degradation of crude oil and/or crudeoil components. The reactive compounds can assist various degradation(e.g., bond-breaking) reactions, leading to a reduction in molecularweight of the exposed material. An exemplary reactive compound is ozone,which can be introduced directly as a gas, or generated in situ viaapplication of a high voltage to an oxygen-containing supply gas (e.g.,oxygen gas, air) or exposure of the oxygen-containing supply gas to anion beam and/or an electron beam. In some embodiments, ion beam exposureof crude oil and/or crude oil components in the presence of a fluid suchas oxygen gas or air can lead to the formation of ozone gas, which alsoassists the degradation of the exposed material.

Prior to and/or following distillation in a refinery, crude oil and/orcomponents thereof can undergo a variety of other refinery processes topurify components and/or convert components into other products. In thefollowing sections, certain additional refinery steps are outlined, anduse of the methods disclosed herein in combination with the additionalrefinery steps will be discussed.

(i) Catalytic Cracking

Catalytic cracking is a widely used refinery process in which heavy oilsare exposed to heat and pressure in the presence of a catalyst topromote cracking (e.g., conversion to lower molecular weight products).Originally, cracking was accomplished thermally, but catalytic crackinghas largely replaced thermal cracking due to the higher yield ofgasoline (with higher octane) and lower yield of heavy fuel oil andlight gases. Most catalytic cracking processes can be classified aseither moving-bed or fluidized bed processes, with fluidized bedprocesses being more prevalent. Process flow is generally as follows. Ahot oil feedstock is contacted with the catalyst in either a feed riserline or the reactor. During the cracking reaction, the formation of cokeon the surface of the catalyst progressively deactivates the catalyst.The catalyst and hydrocarbon vapors undergo mechanical separation, andoil remaining on the catalyst is removed by steam stripping. Thecatalyst then enters a regenerator, where it is reactivated by carefullyburning off coke deposits in air. The hydrocarbon vapors are directed toa fractionation tower for separation into product streams at particularboiling ranges.

Older cracking units (e.g., 1965 and before) were typically designedwith a discrete dense-phase fluidized catalyst bed in the reactorvessel, and operated so that most cracking occurred in the reactor bed.The extent of cracking was controlled by varying reactor bed depth(e.g., time) and temperature. The adoption of more reactive zeolitecatalysts had led to improved modern reactor designs in which thereactor is operated as a separator to separate the catalyst and thehydrocarbon vapors, and control of the cracking process is achieved byaccelerating the regenerated catalyst to a particular velocity in ariser-reactor before introducing it into the riser and injecting thefeedstock into the riser.

The methods disclosed herein can be used before, during, and/or aftercatalytic cracking to treat components of crude oil. In particular, ionbeam exposure (alone, or in combination with other methods) can be usedto pre-treat feedstock prior to injection into the riser, to treathydrocarbons (including hydrocarbon vapors) during cracking, and/or totreat the products of the catalytic cracking process.

Cracking catalysts typically include materials such as acid-treatednatural aluminosilicates, amorphous synthetic silica-aluminacombinations, and crystalline synthetic silica-alumina catalysts (e.g.,zeolites). During the catalytic cracking process, components of crudeoil can be exposed to ions from one or more ion beams to increase theefficiency of these catalysts. For example, the crude oil components canbe exposed to one or more different types of metal ions that improvecatalyst activity by participating in catalytic reactions.Alternatively, or in addition, the crude oil components can be exposedto ions that scavenge typical catalyst poisons such as nitrogencompounds, iron, nickel, vanadium, and copper, to ensure that catalystefficiency remains high. Moreover, the ions can react with coke thatforms on catalyst surfaces to remove the coke (e.g., by processes suchas sputtering, and/or via chemical reactions), either during cracking orcatalyst regeneration.

(ii) Alkylation

In petroleum terminology, alkylation refers to the reaction of lowmolecular weight olefins with an isoparaffin (e.g., isobutane) to formhigher molecular weight isoparaffins. Alkylation can occur at hightemperature and pressure without catalysts, but commercialimplementations typically include low temperature alkylation in thepresence of either a sulfuric acid or hydrofluoric acid catalyst.Sulfuric acid processes are generally more sensitive to temperature thanhydrofluoric acid based processes, and care is used to minimizeoxidation-reduction reactions that lead to the formation of tars andsulfur dioxide. In both processes, the volume of acid used is typicallyapproximately equal to the liquid hydrocarbon charge, and the reactionvessel is pressurized to maintain the hydrocarbons and acid in a liquidstate. Contact times are generally from about 10 to 40 minutes, withagitation to promote contact between the acid and hydrocarbon phases. Ifacid concentrations fall below about 88% by weight sulfuric acid orhydrofluoric acid, excessive polymerization can occur in the reactionproducts. The use of large volumes of strong acids makes alkylationprocesses expensive and potentially hazardous.

The methods disclosed herein can be used before, during, and/or afteralkylation to treat components of crude oil. In particular, ion beamexposure (alone, or in combination with other methods) during alkylationcan assist the addition reaction between olefins and isoparaffins. Insome embodiments, ion beam exposure of the crude oil components canreduce or even eliminate the need for sulfuric acid and/or hydrofluoricacid catalysts, reducing the cost and the hazardous nature of thealkylation process. The types of ions, the number of ion beam exposures,the exposure duration, and the ion beam current can be adjusted topreferentially encourage 1+1 addition reactions between the olefins andisoparaffins, and to discourage extended polymerization reactions fromoccurring.

(iii) Catalytic Reforming and Isomerization

In catalytic reforming processes, hydrocarbon molecular structures arerearranged to form higher-octane aromatics for the production ofgasoline; a relatively minor amount of cracking occurs. Catalyticreforming primarily increases the octane of motor gasoline.

Typical feedstocks to catalytic reformers are heavy straight-runnaphthas and heavy hydrocracker naphthas, which include paraffins,olefins, naphthenes, and aromatics. Paraffins and naphthenes undergo twotypes of reactions during conversion to higher octane components:cyclization, and isomerization. Typically, paraffins are isomerized andconverted, to some extent, to naphthenes. Naphthenes are subsequentlyconverted to aromatics. Olefins are saturated to form paraffins, whichthen react as above. Aromatics remain essentially unchanged.

During reforming, the major reactions that lead to the formation ofaromatics are dehydrogenation of naphthenes and dehydrocyclization ofparaffins. The methods disclosed herein can be used before, during,and/or after catalytic reformation to treat components of crude oil. Inparticular, ion beam exposure (alone, or in combination with othermethods) can be used to initiate and sustain dehydrogenation reactionsof naphthenes and/or dehydrocyclization reactions of paraffins to formaromatic hydrocarbons. Single or multiple exposures of the crude oilcomponents to one or more different types of ions can be used to improvethe yield of catalytic reforming processes. For example, in certainembodiments, dehydrogenation reactions and/or dehydrocyclizationreactions proceed via an initial hydrogen abstraction. Exposure tonegatively charged, basic ions can increase the rate at which suchabstractions occur, promoting more efficient dehydrogenation reactionsand/or dehydrocyclization reactions. In some embodiments, isomerizationreactions can proceed effectively in acidic environments, and exposureto positively charged, acidic ions (e.g., protons) can increase the rateof isomerization reactions.

Catalysts used in catalytic reformation generally include platinumsupported on an alumina base. Rhenium can be combined with platinum toform more stable catalysts that permit lower pressure operation of thereformation process. Without wishing to be bound by theory, it isbelieved that platinum serves as a catalytic site for hydrogenation anddehydrogenation reactions, and chlorinated alumina provides an acid sitefor isomerization, cyclization, and hydrocracking reactions. In general,catalyst activity is reduced by coke deposition and/or chloride lossfrom the alumina support. Restoration of catalyst activity can occur viahigh temperature oxidation of the deposited coke, followed bychlorination of the support.

In some embodiments, ion beam exposure can improve the efficiency ofcatalytic reformation processes by treating catalyst materials duringand/or after reformation reactions occur. For example, catalystparticles can be exposed to ions that react with and oxidize depositedcoke on catalyst surfaces, removing the coke and maintaining/returningthe catalyst in/to an active state. The ions can also react directlywith undeposited coke in the reformation reactor, preventing depositionon the catalyst particles. Moreover, the alumina support can be exposedto suitably chosen ions (e.g., chlorine ions) to re-chlorinate thesurface of the support. By maintaining the catalyst in an active statefor longer periods and/or scavenging reformation by-products, ion beamexposure can lead to improved throughput and/or reduced operating costsof catalytic reformation processes.

(iv) Catalytic Hydrocracking

Catalytic hydrocracking, a counterpart process to ordinary catalyticcracking, is generally applied to crude oil components that areresistant to catalytic cracking. A catalytic cracker typically receivesas feedstock more easily cracked paraffinic atmospheric and vacuum gasoils as charge stocks. Hydrocrackers, in contrast, typically receivearomatic cycle oils and coker distillates as feedstock. The higherpressures and hydrogen atmosphere of hydrocrackers make these componentsrelatively easy to crack.

In general, although many different simultaneous chemical reactionsoccur in a catalytic hydrocracker, the overall chemical mechanism isthat of catalytic cracking with hydrogenation. In general, thehydrogenation reaction is exothermic and provides heat to the(typically) endothermic cracking reactions; excess heat is absorbed bycold hydrogen gas injected into the hydrocracker. Hydrocrackingreactions are typically carried out at temperatures between 550 and 750°F., and at pressures of between 8275 and 15,200 kPa. Circulation oflarge quantities of hydrogen with the feedstock helps to reduce catalystfouling and regeneration. Feedstock is typically hydrotreated to removesulfur, nitrogen compounds, and metals before entering the firsthydrocracking stage; each of these materials can act as poisons to thehydrocracking catalyst.

Most hydrocracking catalysts include a crystalline mixture ofsilica-alumina with a small, relatively uniformly distributed amount ofone or more rare earth metals (e.g., platinum, palladium, tungsten, andnickel) contained within the crystalline lattice. Without wishing to bebound by theory, it is believed that the silica-alumina portion of thecatalyst provides cracking activity, and the rare earth metals promotehydrogenation. Reaction temperatures are generally raised as catalystactivity decreases during hydrocracking to maintain the reaction rateand product conversion rate. Regeneration of the catalyst is generallyaccomplished by burning off deposits which accumulate on the catalystsurface.

The methods disclosed herein can be used before, during, and/or aftercatalytic hydrocracking to treat components of crude oil. In particular,ion beam exposure (alone, or in combination with other methods) can beused to initiate hydrogenation and/or cracking processes. Single ormultiple exposures of the crude oil components to one or more differenttypes of ions can be used to improve the yield of hydrocracking bytailoring the specific exposure conditions to various process steps. Forexample, in some embodiments, the crude oil components can be exposed tohydride ions to assist the hydrogenation process. Cracking processes canbe promoted by exposing the components to reactive ions such as protonsand/or carbon ions.

In certain embodiments, ion beam exposure can improve the efficiency ofhydrocracking processes by treating catalyst materials during and/orafter cracking occurs. For example, catalyst particles can be exposed toions that react with and oxidize deposits on catalyst surfaces, removingthe deposits and maintaining/returning the catalyst in/to an activestate. The crude oil components can also be exposed to ions thatcorrespond to some or all of the metals used for hydrocracking,including platinum, palladium, tungsten, and nickel. This exposure tocatalytic ions can increase the overall rate of the hydrocrackingprocess.

(v) Other Processes

A variety of other processes that occur during the course of crude oilrefining can also be improved by, or supplanted by, the methodsdisclosed herein. For example, the methods disclosed herein, includingion beam treatment of crude oil components, can be used before, during,and/or after refinery processes such as coking, thermal treatments(including thermal cracking), hydroprocessing, and polymerization toimprove the efficiency and overall yields, and reduce the wastegenerated from such processes.

Other embodiments are within the scope of the following claims.

What is claimed is:
 1. A method comprising: processing a cellulosic orlignocellulosic material that has been exposed to an ion beam generatedby an accelerator, to produce a product or intermediate; the ion beamproviding a radiation dose of greater than 10 Mrad to the cellulosic orlignocellulosic material.
 2. The method of claim 1, where in theaccelerator is selected from the group consisting of: a pulsedaccelerator, a continuous wave accelerator, a LINAC accelerator, and aDC accelerator.
 3. The method of claim 2, wherein the DC acceleratordevice comprises a DC insulated core transformer (ICT) type system. 4.The method of claim 1, wherein the radiation dose is provided at a doserate of about 1 Mrad/s to about 10 Mrad/s.
 5. The method of claim 1,wherein the exposure provides a dose rate of at least 1 Mrad/s.
 6. Themethod of claim 1, wherein the cellulosic or lignocellulosic material issaccharified utilizing an enzyme.
 7. The method of claim 1, wherein theaccelerator has an output power of at least 50 kW.
 8. The method ofclaim 1, wherein the radiation dose is between about 10 and 50 Mrad. 9.The method of claim 1, wherein the accelerator has an output power of atleast 75 kW.
 10. The method of claim 1, wherein the accelerator has anoutput power of at least 100 kW.
 11. The method of claim 1, wherein theradiation dose is between 10 and 100 Mrad.
 12. The method of claim 1,wherein the radiation dose is between 10 and 50 Mrad.
 13. The method ofclaim 1, further comprising converting the saccharified cellulosic orlignocellulosic material into a product.
 14. The method of claim 13,wherein the product is an alcohol.
 15. The method of claim 1, whereinthe cellulosic or lignocellulosic material is selected from the groupconsisting of: plant waste, agricultural waste, forestry waste, yardwaste, animal waste, municipal waste, sewage, synthetic celluloses,paper, paper products, paper waste, wood, wood pulp, particle board,sawdust, silage, alfalfa, hay, grasses, switchgrass, bagasse, cotton,jute, hemp, flax, bamboo, sisal, abaca, straw, rice straw, barley straw,wheat straw, canola straw, oat straw, rice hulls, oat hulls, corn cobs,corn fiber, corn stover, soybean stover, rice hulls, coconut hair,cotton, microbial biomass, seaweed, algae, plankton, bacteria, yeast,and mixtures of these.
 16. The method of claim 15, wherein thecellulosic or lignocellulosic material is corn cob.
 17. The method ofclaim 15, wherein the cellulosic or lignocellulosic material is cornstover.
 18. The method of claim 1, wherein the cellulosic orlignocellulosic material also comprises a starch or a starchy material.19. The method of claim 1, further comprising mechanically treating thecellulosic or lignocellulosic material.
 20. The method of claim 19,wherein the cellulosic or lignocellulosic material is treated beforeirradiation.
 21. The method of claim 19, wherein the cellulosic orlignocellulosic material is treated after irradiation.
 22. The method ofclaim 19, wherein the mechanical treatment reduces the particle size ofthe material.
 23. The method of claim 19, wherein the mechanicaltreatment is selected from the group consisting of cutting, grinding,shearing, and chopping.
 24. The method of claim 19, wherein themechanical treatment reduces the particle size of the material to anaverage of 1.59 mm or less.
 25. The method of claim 19, wherein themechanical treatment reduces the particle size of the material to anaverage of about 0.2 mm to about 1.5 mm.
 26. The method of claim 1,wherein the accelerator is a LINAC device selected from the groupconsisting of: an L-band LINAC device and an S-band LINAC device. 27.The method of claim 26, wherein the LINAC device has an overall lengthof about 2-4 meters.
 28. The method of claim 1, wherein saccharifyingthe irradiated cellulosic or lignocellulosic material yields a sugar,and wherein the method further comprises converting the sugar into aproduct.
 29. The method of claim 28, wherein converting the sugar into aproduct comprises fermenting the sugar to produce an alcohol.
 30. Themethod of claim 29, wherein the product comprises the alcohol.
 31. Themethod of claim 30, wherein the alcohol is selected from the groupconsisting of: ethanol, methanol, propanol, isopropanol, butanol,ethylene glycol, propylene glycol, 1,4-butane diol, glycerin, andmixtures thereof.
 32. The method of claim 31, wherein the butanol isselected from the group consisting of: n-butanol, sec-butanol, andt-butanol.
 33. The method of claim 1, wherein the ion beam effects achain scission reaction effective to provide a yield increase in theproduct or intermediate extracted at a temperature and pressureeffective to extract the product or intermediate.
 34. The method ofclaim 1, wherein the accelerator has a filter to remove undesiredspecies.
 35. The method of claim 1, wherein the ion beam comprisespositively charged ions.
 36. The method of claim 1, wherein the ion beamcomprises at least one of protons, carbon ions, oxygen ions, and noblegas ions.
 37. The method of claim 1, wherein the ion beam comprises atleast one of platinum ions, palladium ions, rhenium ions, iridium ions,ruthenium ions, aluminum ions, nickel ions, and osmium ions.
 38. Themethod of claim 1, further comprising exposing the material to anelectron beam.
 39. The method of claim 1, further comprising exposingthe material to a reactive gas during exposure of the material to theion beam.
 40. The method of claim 39, wherein the reactive gas comprisesozone.
 41. The method of claim 1, wherein exposing the material to theion beam comprises exposing the material to a first type of ions from afirst ion beam, and exposing the material from a second type of ionsfrom a second ion beam.
 42. The method of claim 41, wherein the firstand second types of ions have different charges.
 43. The method of claim41, wherein the first and second types of ions have different masses.44. The method of claim 1, wherein during exposure to the ion beam thematerial is flowing.
 45. The method of claim 1, wherein the ion beamcomprises charged particles having an energy of 10 MeV/u or more. 46.The method of claim 1, wherein exposure to the ion beam breaks chemicalbonds in at least a portion of the cellulosic or lignocellulosicmaterial, leading to a reduction in molecular weight of at least aportion of the cellulosic or lignocellulosic material.
 47. The method ofclaim 1, wherein exposure to the ion beam leads to isomerization of atleast a portion of the cellulosic or lignocellulosic material, theisomerization effecting a reduction in viscosity.
 48. The method ofclaim 1, wherein the ion beam composition is selected to effect theaddition of a functional group to at least a portion of the cellulosicor lignocellulosic material, the functional group effective to increaseionic mobility within the functionalized portion.
 49. The method ofclaim 1, wherein the ion beam comprises hydride ions.
 50. The method ofclaim 1, wherein the chain scission reaction comprises a ring-openingchain scission reaction.
 51. The method of claim 1, wherein the ion beamcomprises ions that behave chemically as Lewis acid moieties or Lewisbase moieties when exposed to the cellulosic or lignocellulosicmaterial.